Methods of reducing level of one or more impurities in a sample during protein purification

ABSTRACT

The present invention provides novel and improved protein purification processes which incorporate certain types of carbonaceous materials and result in effective and selective removal of certain undesirable impurities without adversely affecting the yield of the desired protein product.

RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 14/747,029, filed Jun. 23, 2015 which is acontinuation application of U.S. patent application Ser. No. 13/565,463,filed on Aug. 2, 2012, patented as U.S. Pat. No. 9,096,648 on Aug. 4,2015, which claims the benefit of priority of U.S. Provisional PatentApplication Nos. 61/666,240, filing date Jun. 29, 2012, and U.S.Provisional Patent Application No. 61/575,349, filing date Aug. 19,2011, each of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to improved chromatography methods andmethods of reducing the level of one or more impurities during proteinpurification.

BACKGROUND

Chromatography is a dominant purification technique in the purificationof biological materials, e.g., monoclonal antibodies.

Commonly used chromatography methods include one or more of affinitychromatography media, ion exchange chromatography media, hydrophobicinteraction, hydrophilic interaction, size exclusion and mixed mode(i.e., combination of various chromatography interactions)chromatography. For example, for the purification of monoclonalantibodies, a typical purification process includes an initial Protein Aaffinity capture step followed by one or more ion exchange polishingsteps, the purpose of which is to reduce the level of one or moreimpurities such as, e.g., host cell protein (HCP). Further, otherchromatography techniques, such as: bind and elute hydrophobicinteraction chromatography (HIC); flow-through hydrophobic interactionchromatography (FTHIC); flow-through anion-exchange chromatography(AEX); weak partitioning chromatography with cation-exchange,anion-exchange, or hydrophobic interaction reins; mixed modechromatography techniques, e.g., bind and elute weak cation and anionexchange, bind and elute hydrophobic and ion exchange interaction andflow-through hydrophobic and ion exchange mixed mode interaction (FTMM),both of which can utilize resins such as Capto™ Adhere, Capto™ MMC, HEAHypercel™, PPA Hypercel™, may be used. Additionally, hydrophobic chargeinduction (HCl) chromatography along with others and combinations ofvarious techniques can be used for polishing.

Although, chromatography offers many advantages for protein purificationon a smaller scale, on a large scale, packing of chromatography columnsis not only labor and time intensive but also expensive. Further,fouling of chromatography columns is a common problem, resulting in auser having to dispose off columns, which is undesirable, especially dueto the high cost of chromatography resins.

Recently, there has been a noticeable trend in the industry to try andreduce the number of steps in protein purification processes. Also, useof techniques for obtaining a higher expression titer using bioreactorsis a rising trend in the industry. The combination of these two trendshas resulted in more product being loaded onto a column, therebyresulting in increased burden of fairly expensive chromatography mediaas well as lower product purity, both of which are undesirable.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the surprising andunexpected discovery that certain materials (e.g., carbonaceous materialsuch as activated carbon) can be incorporated into chromatography columnbased protein purification processes in a flow-through mode, resultingin reducing the burden of chromatography columns, and consequentlyincreasing the life span of chromatography columns.

Further, the present invention is based on the surprising and unexpecteddiscovery that carbonaceous material (e.g., activated carbon) can beused either upstream or downstream of a capture chromatography step toreduce the level of one or more impurities. In some embodimentsaccording to the claimed methods, a sample is contacted with acarbonaceous material before a cation exchange (CEX) chromatographystep. In other embodiments, a cation exchange (CEX) chromatography stepis used before contacting a sample with a carbonaceous material. In yetother embodiments, a sample is contacted with a carbonaceous materialafter a Protein A affinity capture step. Alternatively, the Protein Aaffinity chromatography step may be used after contacting the samplewith a carbonaceous material. In certain embodiments, the Protein Aaffinity capture step may be followed by an anion exchange (AEX)flow-through chromatography step and with or without a CEXchromatography bind/elute step. In still other embodiments, thecarbonaceous material may be used after a non-affinity capture step(e.g., using CEX bind and elute chromatography as a capture step) and isfollowed by an AEX chromatography step.

Further, the present invention provides chromatography based proteinpurification processes which include fewer steps than conventionalprocesses.

In one aspect according to the present invention, a method for reducingthe burden of one or more chromatography columns is provided. In someembodiments, such a method comprises contacting a sample comprising aprotein of interest and one or more impurities in a flow-through modewith one of: (i) a carbonaceous material; (ii) a combination of acarbonaceous material and CEX media; (iii) a combination of acarbonaceous material and AEX media; (iv) a combination of acarbonaceous material and mixed mode media; (v) a combination of acarbonaceous material and HIC media, and (vi) a combination of acarbonaceous material and CEX, AEX and mixed mode media, prior tocontacting the sample with one or more chromatography columns containingaffinity media, AEX media, CEX media, HIC media or mixed-mode media,thereby to reduce the burden of one or more chromatography columns.

In another aspect according to the claimed methods, a method of reducingthe level of one or more impurities in a sample containing a protein ofinterest and the one or more impurities is provided, where the methodcomprises the steps of: (i) contacting a sample comprising a protein ofinterest and one or more impurities with one or more chromatographycolumns containing affinity media, AEX media, CEX media, HIC media ormixed-mode media, under conditions such that the protein of interestbinds to the column; (ii) obtaining a first eluate of the sample; (iii)contacting the first eluate in flow-through mode with one of: (a) acarbonaceous material; and (b) a combination of a carbonaceous materialand one or more of CEX media, AEX media, mixed mode media and HIC media;and (iv) obtaining a second eluate of the sample; where the secondeluate comprises lower or reduced level of one or more impuritiesrelative to the level of one or more impurities in the first eluate.

In yet another aspect, a method of reducing the level of one or moreimpurities in a sample comprising a protein of interest and one or moreimpurities is provided, the method comprising the steps of: (i)contacting a sample comprising a protein of interest and one or moreimpurities with a chromatography column containing affinity media; (ii)obtaining a first eluate of the sample; (iii) contacting the firsteluate in flow-through mode with a carbonaceous material; (iv) obtaininga second eluate of the sample; (v) contacting the second eluate with ananion exchange chromatography media; and (vi) obtaining a third eluateof the sample, wherein the third eluate comprises lower or reduced levelof one or more impurities relative to the level of one or moreimpurities when the first eluate is not contacted with the carbonaceousmaterial.

In some embodiments, such a method includes a CEX bind and elutechromatography step after the affinity capture step and beforecontacting the sample with an anion exchange chromatography media, whichin some embodiments is a membrane adsorber. In some embodiments, amethod according to the claimed invention obviates the need for furtherchromatography steps, e.g., a bind and elute CEX chromatography stepused after the affinity capture step. Exemplary commercially availableanion exchange chromatography media are membrane adsorbers such asChromaSorb™ (MILLIPORE CORPORATION, Billerica, Mass., USA), Mustang Q(PALL CORPORATION, Port Washington, N.Y., USA), Sartobind Q (SARTORIUSSTEDIM, Germany), as well as bead media such as Q Sepharose FF (GEHEALTHCARE, Philadelphia, Pa., USA).

In some embodiments, methods according to the claimed invention employnon-column based chromatography steps.

In yet another aspect, a method of reducing the level of one or moreimpurities in a sample comprising a protein of interest is provided, themethod comprising the steps of: (i) obtaining a protein phase comprisingthe protein of interest; (ii) reconstituting the protein phasecomprising the protein of interest using a suitable buffer, thereby toobtain a reconstituted protein solution; (iii) contacting thereconstituted protein solution with a carbonaceous material inflow-through mode; (iv) obtaining a first eluate comprising the proteinof interest; (v) contacting the first eluate with an anion exchangechromatography media; and (vi) obtaining a second eluate comprising theprotein of interest, wherein the second eluate comprises a lower orreduced level of one or more impurities relative to the level of one ormore impurities when the reconstituted protein solution from (iii) isnot contacted with the carbonaceous material.

In some embodiments, such a method obviates the need for any bind andelute chromatography steps, e.g., a bind and elute affinity or CEXchromatography steps.

In some methods according to the present invention, the protein phase isobtained using one or more methods selected from the group consisting ofprecipitation, flocculation, crystallization, column chromatography, useof a soluble small molecule, use of a polymeric ligand, or use of asuspended chromatography media.

In some embodiments, combination of a carbonaceous material and one ormore of AEX media, CEX media, HIC media and mixed media entails mixingthe carbonaceous material with one or more of such media. In otherembodiments, combination of a carbonaceous material and one or more ofAEX media, CEX media, HIC media and mixed media entails using differentmaterials in the combination in tandem.

In various embodiments according to the methods of the presentinvention, the affinity media is selected from Protein A or Protein G.

In some embodiments, the protein of interest is an antibody or an Fcregion containing protein. In some embodiments, the antibody is amonoclonal antibody. In other embodiments, the antibody is a polyclonalantibody.

In some embodiments, the sample comprises a cell culture feed.

In some embodiments, the sample is a clarified cell culture feed.

In some embodiments, the clarified cell culture feed is obtained viadepth filtration and/or centrifugation.

In some embodiments, the clarified cell culture is obtained viaprecipitation with a salt, an acid, a polymer, or a stimulus responsivepolymer.

In various embodiments, the carbonaceous material used in the methodsaccording to the claimed invention is activated carbon. In someembodiments, activated carbon comprises activated charcoal.

In some embodiments, the combination of a carbonaceous material and oneor more of CEX media, AEX media, mixed mode media and HIC mediacomprises a mixture of activated carbon and one or more of CEX resin,AEX resin, mixed mode resin and HIC resin. In some embodiments, such amixture is packed into a chromatography column. In other embodiments,the mixture is packed into a disc. In still other embodiments, themixture is packed into a pod, cartridge or a capsule.

In some embodiments, activated carbon is packed into a chromatographycolumn. In other embodiments, activated carbon is packed in a sealeddisposable device such as Millistak+® Pod. In yet other embodiments,activated carbon is packed in a cartridge or a capsule.

In some embodiments, activated carbon is impregnated into a porousmaterial, e.g. activated carbon is incorporated into porous fibrousmedia. The porous material may be contained within a column, a disc, aMillistak+® Pod, a cartridge or a capsule. In some embodiments,activated carbon is packed into a cellulose media.

In a particular embodiment, the AEX media is a membrane having a surfacecoating comprising one or more polymeric primary amines or copolymersthereof.

In some embodiments, a sample comprising a protein of interest and oneor more impurities is contacted with activated carbon prior tosubjecting the sample to an affinity capture step. In other embodiments,the sample is contacted with activated carbon after the affinity capturestep.

In various methods according to the claimed invention, the loss in yieldof the protein of interest using a process which employs activatedcarbon is less than 20% of the total protein amount. In other words,processes according to the claimed invention result in 80% or greateryield of protein of interest, where 100% is the total protein amount. Ina further embodiment, the loss of yield of the protein of interest usinga process which employs activated carbon is less than 10%. In otherwords, processes according to the claimed invention result in 90% orgreater yield of protein of interest, where 100% is the total proteinamount.

In a particular embodiment, activated carbon is used as part of aflow-through purification process step or unit operation in a method forpurifying a target molecule (e.g., an Fc region containing protein or anantibody) from a sample (e.g., an eluate such as a Protein A eluaterecovered from a bind and elute chromatography capture process stepperformed prior to the flow-through purification step). In such aflow-through purification process step or unit operation, the eluatefrom a bind and elute chromatography step (e.g., a Protein A affinitycolumn) flows through activated carbon followed by an AEX media followedby a CEX media and followed by a virus filter, as depicted in FIG. 19 .In some embodiments, a solution change (e.g., pH change) is performedbetween the AEX step and the CEX step, where the solution employs anin-line static mixer and/or a surge tank. In some embodiments, theflow-through purification process step or unit operation employingactivated carbon, as described herein, is part of a continuous processfor purifying a target molecule, where the flow-through purificationstep is in fluid communication with a process step upstream (e.g., abind and elute chromatography capture step) and a process stepdownstream (e.g., a formulation step) of the flow-through purificationprocess step, thereby enabling the liquid sample to flow through theprocess continuously.

In a particular embodiment, the entire flow-through process step or unitoperation employs a single skid (i.e., a control/monitoring equipment).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a bar graph demonstrating the results of an experiment tomeasure IgG yield in a flow-through eluate of a null CHO-S feed withadded polyclonal IgG for each of the various commercially availableadsorptive media that were evaluated, i.e., activated carbon (AC); anagarose cation exchange resin, SP Sepharose™ Fastflow (SPFF); apolymeric cation exchange resin, ProRes™-S; an agarose anion exchangeresin, Q Sepharose™ (QFF); and an agarose HIC resin, Phenyl Sepharose™ 6Fastflow (ph FF). As demonstrated in FIG. 1 , except for the HIC resinwhich shows up to ˜5% loss in IgG yield, all other media screeneddemonstrated no detectable yield loss.

FIG. 2 depicts a bar graph demonstrating the results of an experiment tomeasure the amount of UV 280 nm active species in a flow-through eluateof a null CHO-S feed with added polyclonal IgG, for each of the variouscommercially available adsorptive media that were evaluated, i.e., anactivated carbon, Nuchar® RGC, (AC), SPFF, ProRes™-S, QFF and ph FF aswell as the untreated clarified feed. As demonstrated in FIG. 2 ,activated carbon significantly reduced the amount of colored speciescompared with the other adsorptive media.

FIG. 3 depicts a bar graph demonstrating the results of an experiment tomeasure the concentration of host cell protein (HCP) in a flow-througheluate of a null CHO-S feed with added polyclonal IgG, for each of thecommercially available adsorptive media listed above, as well as theuntreated clarified feed. The HCP concentration was measured in ng/mLusing a Cygnus CHO-CM HCP ELISA kit. As demonstrated in FIG. 3 , all themedia screened, including activated carbon, removed HCP to some extent.However, the cationic resins SPFF and ProRes™-S, removed HCP mosteffectively.

FIG. 4 depicts a bar graph demonstrating the results of an experiment tomeasure the DNA concentration in a flow-through eluate of a null CHO-Sfeed with added polyclonal IgG, for each of the media listed above, aswell as the untreated clarified feed. DNA concentration (μg/mL) wasmeasured using a PicoGreen assay. As demonstrated in FIG. 4 , each ofthe media removes DNA to some extent.

However, the anion exchange media removes DNA most effectively, followedby activated carbon.

FIG. 5 depicts an x-y scatter plot demonstrating the results of anexperiment to measure the concentration of IgG in a flow-through eluateof a null CHO-S feed with added polyclonal IgG and Herring sperm DNA,for each of the media evaluated listed above, at every 10 CV of feedloaded, up to 100 CV, including the untreated clarified feed. Columnvolume (CV) is shown on the x-axis and IgG concentration in mg/mL isshown on the y-axis. All of the media evaluated, including AC, SPFF,ProRes™-S and QFF, showed no significant loss of IgG up to a loading of100 CV of untreated clarified feed.

FIG. 6 depicts an x-y scatter plot demonstrating the results of anexperiment to measure the UV active species peak area (corresponding tothe quantity of UV active species) in a flow-through eluate of a nullCHO-S feed added polyclonal IgG and Herring sperm DNA, for each of themedia evaluated, at every 10 CV of feed loaded, up to 100 CV, includingthe untreated clarified feed. Column volume (CV) is shown on the x-axisand UV active species peak area in the flow-through of Protein Aanalytical column is shown on the y-axis. Of all the materialsevaluated, AC removed more than 70% of the UV active species throughoutthe 100 CV; QFF removed about 10% of UV active species throughout the100 CV; and the two cation exchange resins, SPFF and ProRes™-S removedminimal amount of UV active species.

FIG. 7 depicts an x-y scatter plot demonstrating the results of anexperiment to measure the host cell protein (HCP) concentration in aflow-through eluate of a null CHO-S feed with added polyclonal IgG andHerring sperm DNA, for each of the media evaluated and listed above, atevery 10 CV of feed loaded up to 100 CV, including the untreatedclarified feed. Column volume (CV) is shown on the x-axis and HCPconcentration in ng/mL is shown on the y-axis. SPFF and ProRes S removedthe most HCP throughout the 100 CV. QFF removed some HCP but brokethrough quickly. For this specific feed which had a high concentrationof DNA, activated carbon removed the least amount of HCP.

FIG. 8 depicts an x-y scatter plot demonstrating the result of anexperiment to measure DNA concentration in a flow-through eluate of anull CHO-S feed with added polyclonal IgG and Herring sperm DNA, foreach of the media evaluated and listed above, at every 10 CV of feedloaded up to 100 CV, including the untreated clarified feed. Columnvolume (CV) is shown on the x-axis and DNA concentration in μg/mL isshown on the y-axis. Each of the media evaluated, including AC, SPFF,ProRes™-S and QFF, removed DNA throughout the 100 CV, however, todifferent degrees.

FIG. 9 is a schematic of the different exemplary modes of operationwhich may be used for impurity removal. The flow chart on the leftdepicts a representative experiment where the untreated clarified feedis loaded onto a column containing AC, followed by a column containingSPFF, ProRes™-S or QFF media. The flow chart in the middle depicts arepresentative experiment where the untreated clarified feed is loadedonto a column containing AC, followed by a column containing SPFF orProRes™-S and then followed by QFF. The flow chart on the right depictsa representative experiment where untreated clarified feed is loadedonto a column containing a 1:1 (v/v) mixture of AC and SPFF; or a 1:1(v/v) mixture of AC and ProRes™-S; or a 1:1:1 (v/v/v) mixture of AC andProRes™-S and QFF.

FIG. 10 depicts a bar graph demonstrating UV active species peak area(which corresponds to the quantity of UV active species) in aflow-through eluate of a null CHO-S feed with added polyclonal IgG, foreach of the material combinations shown in FIG. 9 , including untreatedclarified feed. Activated carbon and mixtures which contain activatedcarbon significantly reduced the UV active species. In the cases whereactivated carbon and an anion exchange resin were both used in aprocess, either when used sequentially or as a mixture, more UV activespecies were removed, demonstrating a synergetic effect of differentmaterials.

FIG. 11 depicts a bar graph which shows the host cell protein (HCP)concentration in a flow-through eluate of a null CHO-S feed with addedpolyclonal IgG, for each of the material combinations shown in FIG. 9 ,including untreated clarified feed. All materials removed HCP to somedegree; however, when activated carbon was used in a process along witha cationic resin, such as, SPFF or ProRes™-5, or with an anionic resin,such as, QFF, either when used sequentially with a resin or as a mixturewith a resin, removed HCP most effectively, indicating a synergeticeffect of different materials.

FIG. 12 depicts a bar graph which shows the DNA concentration in aflow-through eluate of a null CHO-S feed with added polyclonal IgG, foreach of the material combinations shown in FIG. 9 , including untreatedclarified feed. All materials removed DNA to some degree; however, whenactivated carbon was used either sequentially with QFF or used as amixture with QFF, it was most effective in DNA removal compared to theother materials and combinations evaluated.

FIG. 13 depicts a bar graph demonstrating the results of an experimentto measure IgG yield in a flow-through eluate of a Protein A columnelution pool for each of the materials evaluated i.e., AC; SPFF; QFF; phFF, as well as two material combinations, a 1:1:1 (v/v/v) mixture ofAC/SPFF/QFF, and a 1:1:1 (v/v/v) mixture of PhFF/SPFF/QFF. The feed forthe flow-through eluate of different materials evaluated was a Protein Acolumn elution pool generated using Prosep Ultra Plus Protein A resinfrom a null CHO-S feed with added polyclonal IgG. All materials screenedshowed higher than 80% yield.

FIG. 14 depicts a bar graph demonstrating the results of an experimentto measure host cell protein (HCP) concentration in a flow-througheluate of a Protein A column elution pool for each of the materialsevaluated, i.e., AC; SPFF; QFF; ph FF, as well as two materialcombinations, a 1:1:1 (v/v/v) mixture of AC/SPFF/QFF and a 1:1:1 (v/v/v)mixture of PhFF/SPFF/QFF. The feed for flow-through eluate of differentmaterials evaluated was a Protein A column elution pool generated usingProsep Ultra Plus Protein A resin from a null CHO-S feed with addedpolyclonal IgG. All materials or material mixtures removed certainamount of HCP from the Protein A elution pool; however, activated carbonand cation exchange resin were the more effective, when used alone. Whenused as a mixture, AC/SPFF/QFF and PhFF/SPFF/QFF removed more HCP thanany single component alone. QFF and PhFF, when used alone, removed theleast amount of HCP.

FIG. 15 a bar graph demonstrating the results of an experiment tomeasure DNA concentration in a flow-through eluate of a Protein A columnelution pool for each of the materials evaluated, i.e., AC; SPFF; QFF;and ph FF, as well as two material combinations, a 1:1:1 mixture ofAC/SPFF/QFF and a 1:1:1 mixture of PhFF/SPFF/QFF. The feed forflow-through eluate of different materials evaluated was a Protein Acolumn elution pool generated using Prosep Ultra Plus Protein A resinfrom a null CHO-S feed with added polyclonal IgG. All material ormaterial mixtures removed certain amount of DNA from Protein A elutionpool with the resin mixtures AC/SPFF/QFF and PhFF/SPFF/QFF showingslight advantage over any single component.

FIG. 16 is a graph demonstrating the results of an experiment to measurethe concentration of HCP relative to that of the product (i.e., amonoclonal antibody) in ppm for the individual fractions of a monoclonalantibody solution, where the solution was captured from clarified cellculture using Protein A chromatography (referred to as Protein A eluate)and was subsequently subjected to three separate flow-throughpurification trains. The first train employed a 0.2 mL ChromaSorb™anion-exchange membrane device composed of 5 layers; the second trainemployed a 1 mL packed column of HD Nuchar activated carbon; and thethird train employed a 1 mL activated carbon column followed by a 0.2 mLChromaSorb™ anion-exchange membrane. Ten 10 mL fractions of the eluatewere collected from each purification train and select fractions wereanalyzed for host cell protein (HCP) and IgG concentration. The X-axisof the graph depicts the end point of collection for the 10 mL fractionin column volumes (CVs) of the eluate from the activated carbon column.The Y-axis of the graph depicts the concentration of HCP relative tothat of the product (i.e., a monoclonal antibody) in ppm for theindividual fractions of an activated carbon eluate. The graphdemonstrates that the flow-through treatment of the affinity capturedeluate with activated carbon alone and in combination with an anionexchange media was unexpectedly effective for the removal of impuritiesfrom the monoclonal antibody solution.

FIG. 17 is a graph demonstrating the results of an experiment to measurethe concentration of HCP relative to that of the product (i.e., amonoclonal antibody) in ppm for the individual fractions of a monoclonalantibody solution, where the solution was captured from clarified cellculture using cation exchange (CEX) chromatography (referred to as CEXeluate) and was subsequently subjected to purification with a 1 mLpacked column of HD Nuchar activated carbon. Seven 10 mL fractions ofthe eluate were collected, which were analyzed for host cell protein(HCP) and IgG concentration. The X-axis of the graph depicts the endpoint of collection for the 10 mL fraction in eluted volume (mL) of theeluate from the activated carbon column. The Y-axis of the graph depictsthe concentration of HCP relative to that of the product (i.e., amonoclonal antibody) in ppm for the individual fractions. The graphdemonstrates that activated carbon can be used to remove impurities froma variety of different protein solutions.

FIG. 18 is a graph demonstrating the results of an experiment to measurethe concentration of HCP relative to that of the product (i.e., amonoclonal antibody, MAb II) in ppm for the individual fractions of amonoclonal antibody solution, where the solution is captured fromclarified cell culture using a three-column continuousmulti-chromatography chromatography (CMC) system equipped with Protein Acolumns, and subsequently purified with of HD Nuchar activated carbonpacked into a column followed by an anion exchange chromatography device(e.g., ChromaSorb™). The X-axis of the graph depicts the end point offraction collection, measured in the weight of antibody loaded per unitvolume of the anion exchange device (kg/L). The Y-axis of the graphdepicts the concentration of HCP relative to that of the product (i.e.,a monoclonal antibody) in ppm for the individual fractions. The graphdemonstrates that while both activated carbon and ChromaSorb™ remove asignificant portion of HCP when used alone, when used in combination,they increase the purity of the starting solution from 1,370 ppm HCP tounder 10 ppm.

FIG. 19 demonstrates a schematic of the connected flow-throughpurification process step, as described herein. An activated carboncontaining device is connected directly to an anion-exchange device. Theeffluent from the anion-exchange device passes through a static mixer,where an aqueous acid is added to reduce pH, and then goes through acation-exchange flow-through device and a virus filter.

FIG. 20 is a graph depicting the results of an experiment to measure HCPbreakthrough after an anion exchange chromatography device (i.e.,ChromaSorb™) The Y-axis denotes HCP concentration (ppm) and the X-axisdenotes the AEX loading (kg/L).

FIG. 21 is a graph depicting the results of an experiment to measureremoval of MAb aggregates as a function of loading of the virusfiltration device in the flow-through purification process step. TheX-axis denotes the virus filtration loading (kg/m²) and the Y-axisdenotes percentage of MAb aggregates in the sample after virusfiltration.

FIG. 22 is a graph depicting the results of an experiment to measurepressure profiles after depth filter, activated carbon and virusfiltration. The Y-axis denotes pressure (psi) and the X-axis denotestime in hours.

DETAILED DESCRIPTION

The present invention provides novel and improved processes forpurifying a protein of interest from a sample containing the protein ofinterest and one of more impurities.

Activated carbon has previously been used in water purificationprocesses. In addition, activated carbon has been used to remove smallmolecule impurities, such as fatty acids and bilirubin, from serum album(see, e.g., Chen et al., J. Biol. Chem., 242: 173-181 (1967); Nakano etal., Anal Biochem., 129: 64-71 (1983); Nikolaev et al., Int. J. Art.Org., 14:179-185 (1991)). Activated carbon has also been used to removepigments as well as host proteins, proteases, and ribonucleases duringthe purification of plant viruses (see, e.g., Price, Am. J. Botany, 33:45-54 (1946); Corbett, Virology, 15:8-15 (1961); McLeana et al.,Virology, 31: 585-591 (1967).

Accordingly, in general, activated carbon has been reported tonon-specifically bind to molecules in solution (e.g., impurities in awater sample).

The present invention is based, at least in part, on the unexpected andsurprising finding that activated carbon can selectively removepopulations of proteinaceous impurities and DNA, thereby making ituseful in the purification of proteins produced via recombinantexpression in cells.

As demonstrated in the Examples herein, activated carbon can be used forselective removal of host cell protein (HCP) and DNA impurities duringprotein purification processes without significantly affecting the yieldof the target protein. Further, as demonstrated in the Examples setforth herein, when activated carbon is used in a protein purificationprocess in flow-through mode, either alone or in a mixture with one ormore chromatography media of various types, it results in a significantreduction in the level of one or more impurities in the proteincontaining sample as well as reduces the burden of downstreamchromatography columns. Further, in certain instances, activated carbondecreases the number of steps that may be used in a purificationprocess, thereby reducing the overall operational costs and saving time.Further, as demonstrated in the Examples set forth herein, activatedcarbon can be used before or after a capture step, thereby to reduce thelevel of one or more impurities in a sample containing the protein ofinterest.

In some embodiments described herein, activated carbon is used in aflow-through purification step of an overall process for purifying atarget molecule, where the overall process as well as the flow-throughpurification step are performed in a continuous manner.

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

I. Definitions

The term “carbonaceous material,” as used herein, refers to anysubstance composed of carbon or containing carbon. In some embodiments,carbonaceous material used in the methods according to the claimedinvention is active or activated carbon. In some embodiments, activatedcarbon comprises activated charcoal. In some embodiments, activatedcarbon is incorporated into a cellulose media.

The term “active carbon” or “activated carbon,” as used interchangeablyherein, refers to a carbonaceous material which has been subjected to aprocess to enhance its pore structure. Activated carbons are poroussolids with very high surface areas. They can be derived from a varietyof sources including coal, wood, coconut husk, nutshells, and peat.Activated carbon can be produced from these materials using physicalactivation involving heating under a controlled atmosphere or chemicalactivation using strong acids, bases, or oxidants. The activationprocesses produce a porous structure with high surface areas that giveactivated carbon high capacities for impurity removal. Activationprocesses can be modified to control the acidity of the surface.

Typical activation processes involve subjecting a carbon source, suchas, resin wastes, coal, coal coke, petroleum coke, lignites, polymericmaterials, and lignocellulosic materials including pulp and paper,residues from pulp production, wood (like wood chips, sawdust, and woodflour), nut shell (like almond shell and coconut shell), kernel, andfruit pits (like olive and cherry stones) to a thermal process (e.g.,with an oxidizing gas) or a chemical process (e.g., with phosphoric acidor metal salts, such as zinc chloride). An exemplary chemical activationof wood-based carbon with phosphoric acid (H₃PO₄) is disclosed in U.S.Pat. No. Re. 31,093, which resulted in an improvement in the carbon'sdecolorizing and gas adsorbing abilities. Also, U.S. Pat. No. 5,162,286teaches phosphoric acid activation of wood-based material which isparticularly dense and which contains a relatively high (30%) lignincontent, such as nut shell, fruit stone, and kernel. Phosphoric acidactivation of lignocellulose material is also discussed in U.S. Pat. No.5,204,310, as a step in preparing carbons of high activity and highdensity. The teachings of each of the patents listed in this paragraphare incorporated by reference herein in their entirety.

In contrast to most other adsorbing materials, activated carbon isbelieved to interact with molecules using relatively weak van der Waalsor London dispersion forces. Typical commercial activated carbonproducts exhibit a surface area of at least 300 m²/g, as measured by thenitrogen adsorption based Brunauer-Emmett-Teller (“BET”) method, whichis method well known in the art.

Although, active or activated carbon has been previously employed inprocesses for purifying liquids and gases, it has not been previouslyemployed in processes for purifying a recombinantly expressed proteinfrom one or more proteinaceous impurities.

The term “immunoglobulin,” “Ig” or “IgG” or “antibody” (usedinterchangeably herein) refers to a protein having a basicfour-polypeptide chain structure consisting of two heavy and two lightchains, said chains being stabilized, for example, by interchaindisulfide bonds, which has the ability to specifically bind antigen. Theterm “single-chain immunoglobulin” or “single-chain antibody” (usedinterchangeably herein) refers to a protein having a two-polypeptidechain structure consisting of a heavy and a light chain, said chainsbeing stabilized, for example, by interchain peptide linkers, which hasthe ability to specifically bind antigen. The term “domain” refers to aglobular region of a heavy or light chain polypeptide comprising peptideloops (e.g., comprising 3 to 4 peptide loops) stabilized, for example,by (3-pleated sheet and/or intrachain disulfide bond. Domains arefurther referred to herein as “constant” or “variable”, based on therelative lack of sequence variation within the domains of various classmembers in the case of a “constant” domain, or the significant variationwithin the domains of various class members in the case of a “variable”domain. Antibody or polypeptide “domains” are often referred tointerchangeably in the art as antibody or polypeptide “regions”. The“constant” domains of antibody light chains are referred tointerchangeably as “light chain constant regions”, “light chain constantdomains”, “CL” regions or “CL” domains. The “constant” domains ofantibody heavy chains are referred to interchangeably as “heavy chainconstant regions”, “heavy chain constant domains”, “CH” regions or “CH”domains. The “variable” domains of antibody light chains are referred tointerchangeably as “light chain variable regions”, “light chain variabledomains”, “VL” regions or “VL” domains. The “variable” domains ofantibody heavy chains are referred to interchangeably as “heavy chainvariable regions”, “heavy chain variable domains”, “VH” regions or “VH”domains.

Immunoglobulins or antibodies may be monoclonal or polyclonal and mayexist in monomeric or polymeric form, for example, IgM antibodies whichexist in pentameric form and/or IgA antibodies which exist in monomeric,dimeric or multimeric form. Immunoglobulins or antibodies may alsoinclude multispecific antibodies (e.g., bispecific antibodies), andantibody fragments so long as they retain, or are modified to comprise,a ligand-specific binding domain. The term “fragment” or “functionalfragment” of an antibody refers to a part or portion of an antibody orantibody chain comprising fewer amino acid residues than an intact orcomplete antibody or antibody chain. Fragments can be obtained viachemical or enzymatic treatment of an intact or complete antibody orantibody chain. Fragments can also be obtained by recombinant means.When produced recombinantly, fragments may be expressed alone or as partof a larger protein called a fusion protein. Exemplary fragments includeFab, Fab′, F(ab′)2, Fc and/or Fv fragments. Exemplary fusion proteinsinclude Fc fusion proteins.

In a particular embodiment, methods according to the claimed inventionare used for purifying a fragment of an antibody which is an Fc-regioncontaining fragment.

The term “Fc region” and “Fc region containing protein” means that theprotein contains heavy and/or light chain constant regions or domains(CH and CL regions as defined previously) of an immunoglobulin. Proteinscontaining an “Fc region” can possess the effector functions of animmunoglobulin constant domain. An “Fc region” such as CH₂/CH₃ regions,can bind selectively to affinity ligands such as Protein A or functionalvariants thereof. In some embodiments, an Fc region containing proteinspecifically binds Protein A or a functional derivative, variant orfragment thereof. In other embodiments, an Fc region containing proteinspecifically binds Protein G or Protein L, or functional derivatives,variants or fragments thereof.

As discussed above, in some embodiments, a target protein is an Fcregion containing protein, e.g., an immunoglobulin. In some embodiments,an Fc region containing protein is a recombinant protein which includesthe Fc region of an immunoglobulin fused to another polypeptide or afragment thereof.

Generally, an immunoglobulin or antibody is directed against an“antigen” of interest. Preferably, the antigen is a biologicallyimportant polypeptide and administration of the antibody to a mammalsuffering from a disease or disorder can result in a therapeutic benefitin that mammal.

The term “monoclonal antibody” or “Mab,” as used interchangeably herein,refers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies in thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific, being directed against a single antigenic site.Furthermore, in contrast to conventional (polyclonal) antibodypreparations which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature 256:495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).“Monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

Monoclonal antibodies may further include “chimeric” antibodies(immunoglobulins) in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (i.e. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, Fv framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibody maycomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The terms “polynucleotide” and “nucleic acid molecule,” usedinterchangeably herein, refer to polymeric forms of nucleotides of anylength, either ribonucleotides or deoxyribonucleotides. These termsinclude a single-, double- or triple-stranded DNA, genomic DNA, cDNA,RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidinebases, or other natural, chemically or biochemically modified,non-natural or derivatized nucleotide bases. The backbone of thepolynucleotide can comprise sugars and phosphate groups (as maytypically be found in RNA or DNA), or modified or substituted sugar orphosphate groups. In addition, a double-stranded polynucleotide can beobtained from the single stranded polynucleotide product of chemicalsynthesis either by synthesizing the complementary strand and annealingthe strands under appropriate conditions, or by synthesizing thecomplementary strand de novo using a DNA polymerase with an appropriateprimer. A nucleic acid molecule can take many different forms, e.g., agene or gene fragment, one or more exons, one or more introns, mRNA,cDNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracyl, other sugars and linking groups such as fluororibose andthioate, and nucleotide branches. As used herein, “DNA” or “nucleotidesequence” includes not only bases A, T, C, and G, but also includes anyof their analogs or modified forms of these bases, such as methylatednucleotides, internucleotide modifications such as uncharged linkagesand thioates, use of sugar analogs, and modified and/or alternativebackbone structures, such as polyamides

The term “solution,” “composition” or “sample,” as used herein, refersto a mixture of a protein of interest or target protein (e.g., an Fcregion containing protein such as an antibody) and one or moreimpurities. In some embodiments, the sample is subjected to aclarification step prior to being subjected to the methods according tothe claimed invention. In some embodiments, the sample comprises cellculture feed, for example, feed from a mammalian cell culture (e.g., CHOcells). However, samples also encompass non-mammalian expression systemsused for producing a protein of interest.

The term “non-mammalian expression systems” as used herein refers to allhost cells or organisms employed to generate therapeutic proteins, wherethe host cells or organisms are of non-mammalian origin. Non-limitingexamples of non-mammalian expression systems are E. coli and Pichiapastoris.

The term “UV active species” as used herein, refers to the compositionof the flow-through fraction of a clarified cell culture followingsubjecting the culture to a Protein A analytical column, as monitored bya UV spectrophotometer. In some embodiments, the UV spectrophotometermonitors the fraction at 280 nm. This fraction generally consists ofimpurities such as, dyes (such as pH indicators), host cell proteins,DNA, and other cell culture media components that need to be removedfrom the fraction, which also contains the protein of interest (e.g., anantibody). The flow-through impurity peak is integrated manually or by apreset algorithm and is used to quantity the total impurity level.

As used herein, the term “polypeptide” refers generally to peptides andproteins having more than about ten amino acids. The terms “protein ofinterest” and “target protein,” as used interchangeably herein, refer toa protein or polypeptide, including but not limited to, an Fc regioncontaining protein such as an antibody that is to be purified by amethod of the invention, from one or more impurities.

Exemplary polypeptides include, e.g., renin; a growth hormone, includinghuman growth hormone and bovine growth hormone; growth hormone releasingfactor; parathyroid hormone; thyroid stimulating hormone; lipoproteins;α-1-antitrypsin; insulin α-chain; insulin β-chain; proinsulin; folliclestimulating hormone; calcitonin; luteinizing hormone; glucagon; clottingfactors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-αand β; enkephalinase; RANTES (regulated on activation normally T-cellexpressed and secreted); human macrophage inflammatory protein(MIP-1-α); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin α-chain; relaxin β-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as β-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA) (e.g., CTLA-4); inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; ProteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor such as NGF-β.;platelet-derived growth factor (PDGF); fibroblast growth factor such asαFGF and βFGF; epidermal growth factor (EGF); transforming growth factor(TGF) such as TGF-alpha and TGF-β, including TGF-β1, TGF-β2, TGF-β3,TGF-β4, or TGF-β5; insulin-like growth factor-I and -II (IGF-I andIGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factorbinding proteins (IGFBPs); CD proteins such as CD3, CD4, CD8, CD19 CD20,CD34, and CD40; erythropoietin; osteoinductive factors; immunotoxins; abone morphogenetic protein (BMP); an interferon such as interferon-α,-β, and -γ; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, andG-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase;T-cell receptors; surface membrane proteins; decay accelerating factor;viral antigen such as, for example, a portion of the AIDS envelope;transport proteins; homing receptors; addressins; regulatory proteins;integrins such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM; atumor associated antigen such as HER2, HER3 or HER4 receptor; andfragments and/or variants of any of the above-listed polypeptides. Inaddition, a protein or polypeptide of the invention is an antibody,fragment or variant thereof, that binds specifically to any of theabove-listed polypeptides.

The terms “contaminant,” “impurity,” and “debris,” as usedinterchangeably herein, refer to any foreign or objectionable molecule,including a biological macromolecule such as a DNA, an RNA, one or morehost cell proteins (HCP), endotoxins, lipids and one or more additiveswhich may be present in a sample containing the target protein that isbeing separated from one or more of the foreign or objectionablemolecules using a process of the present invention. Additionally, such acontaminant may include any reagent which is used or generated in a stepwhich may occur prior to the purification process, such as leachedprotein A in cases where a protein A affinity chromatography step isemployed.

The terms “Chinese hamster ovary cell protein” and “CHOP” are usedinterchangeably to refer to a mixture of host cell proteins (“HCP”)derived from a Chinese hamster ovary (“CHO”) cell culture. The HCP orCHOP is generally present as an impurity in a cell culture medium orlysate (e.g., a harvested cell culture fluid (“HCCF”)) comprising aprotein of interest such as an antibody or immunoadhesin expressed in aCHO cell). The amount of CHOP present in a mixture comprising a proteinof interest provides a measure of the degree of purity for the proteinof interest. HCP or CHOP includes, but is not limited to, a protein ofinterest expressed by the host cell, such as a CHO host cell. Typically,the amount of CHOP in a protein mixture is expressed in parts permillion relative to the amount of the protein of interest in themixture. It is understood that where the host cell is another cell type,e.g., a mammalian cell besides CHO, an E. coli, a yeast, an insect cell,or a plant cell, HCP refers to the proteins, other than target protein,found in a lysate of the host cell.

The term “parts per million” or “ppm” are used interchangeably herein torefer to a measure of purity of a target protein purified by a method ofthe invention. The units ppm refer to the amount of HCP or CHOP innanograms/milligrams of protein of interest or in milligrams/milliliter(i.e., CHOP ppm=(CHOP ng/ml)/(protein of interest mg/ml), where theproteins are in solution).

The terms “purifying,” “separating,” or “isolating,” as usedinterchangeably herein, refer to increasing the degree of purity of apolypeptide or protein of interest or a target protein from acomposition or sample comprising the protein of interest and one or moreimpurities. Typically, the degree of purity of the protein of interestis increased by removing (completely or partially) at least one impurityfrom the composition. A “purification step” may be part of an overallpurification process resulting in a “homogeneous” composition or sample,which is used herein to refer to a composition or sample comprising lessthan 100 ppm HCP in a composition comprising the protein of interest,alternatively less than 90 ppm, less than 80 ppm, less than 70 ppm, lessthan 60 ppm, less than 50 ppm, less than 40 ppm, less than 30 ppm, lessthan 20 ppm, less than 10 ppm, less than 5 ppm, or less than 3 ppm ofHCP.

The term “protein phase,” as used herein, refers to the part of a samplewhere the concentration of the target protein has been substantiallyincreased relative to the initial concentration of target protein in thesample. The concentration process may involve protein adsorption on asolid porous or non-porous support; protein adsorption at a liquid-airor liquid-gas interface; protein adsorption at the interface between twoimmiscible or partially miscible liquids; protein precipitation as apure component or as a result of complex formation with one or moreother molecules or polymers; or using protein crystallization.

The term “liquid phase” as used herein, refers to that part of a samplewhere the concentration of target protein has been substantially reducedcompared to initial concentration of protein in the sample. The liquidphase can be created at the same time as the protein phase definedabove.

The terms “flow-through process,” “flow-through mode,” and “flow-throughchromatography,” as used interchangeably herein, refer to a productseparation technique in which at least one product in a sample isintended to flow through a chromatographic resin or media, while atleast one potential component binds to the chromatographic resin ormedia.

The sample intended to flow through is generally referred to as the“mobile phase.” The “flow-through mode” is generally an isocraticoperation (i.e., a chromatography process during which the compositionof the mobile phase is not changed). The media used for flow-through isusually pre-equilibrated with the same buffer solution that contains thetarget protein molecule. After purification, the media can be flushedwith additional quantity of the same buffer to increase the productrecovery. In some embodiments, the mobile phase of the “flow-throughmode” is a cell culture feed containing the product of interest. In someinstances, the pH or conductivity of the feed is adjusted in order tomaximize impurity removal using the flow-through process.

In some embodiments according to the claimed methods and as described inthe Examples set forth herein, the methods employ an anion exchange stepwhich is performed in a flow-through mode.

The terms “bind and elute mode” and “bind and elute process,” as usedinterchangeably herein, refer to a product separation technique in whichat least one product contained in a sample binds to a chromatographicresin or media and is subsequently eluted.

The term “chromatography” refers to any kind of technique whichseparates an analyte of interest (e.g., an Fc region containing proteinsuch as an immunoglobulin) from other molecules present in a mixturewhere the analyte of interest is separated from other molecules as aresult of differences in rates at which the individual molecules of themixture migrate through a stationary medium under the influence of amoving phase, or in bind and elute processes.

The term “chromatography resin” or “chromatography media” are usedinterchangeably herein and refer to any kind of porous or non-poroussolid phase which separates an analyte of interest (e.g., an Fc regioncontaining protein such as an immunoglobulin) from other moleculespresent in a mixture. Usually, the analyte of interest is separated fromother molecules as a result of differences in rates at which theindividual molecules of the mixture migrate through a stationary solidphase under the influence of a moving phase, or in bind and eluteprocesses. Non-limiting examples include resins with cationic, anionic,HIC, or mixed mode surface modifications; membranes with cationic,anionic, HIC, or mixed mode surface modifications, woven or non-wovenfibers with cationic, anionic, HIC, or mixed mode surface modifications;and monoliths with cationic, anionic, HIC, or mixed mode surfacemodifications.

The term “affinity separation,” or “affinity purification,” as usedherein, refers to any purification or assaying technique which involvesthe contacting a sample containing a target analyte (e.g., an Fc regioncontaining protein such as an immunoglobulin) with an affinity media(e.g., a solid support carrying on it an affinity ligand known to bindthe analyte such as, for example, e.g., Protein A or a variant thereof)known to bind the target analyte.

The terms “affinity chromatography” and “protein affinitychromatography,” as used interchangeably herein, refer to a proteinseparation technique in which a target protein (e.g., an Fc regioncontaining protein of interest or an antibody) is specifically bound toa ligand which is specific for the target protein. In some embodiments,such a ligand is Protein A or Protein G or a functional variant thereof,which is covalently attached to a chromatographic solid phase materialand is accessible to the target protein in solution as the solutioncontacts the chromatographic solid phase material. The target proteingenerally retains its specific binding affinity for the ligand duringthe chromatographic steps, while other solutes and/or proteins in themixture do not bind appreciably or specifically to the ligand. Bindingof the target protein to the immobilized ligand allows contaminatingproteins or protein impurities to be passed through the chromatographicmedium while the target protein remains specifically bound to theimmobilized ligand on the solid phase material. The specifically boundtarget protein is then removed in active form from the immobilizedligand under suitable conditions (e.g., low pH, high pH, high salt,competing ligand etc.), and passed through the chromatographic columnwith the elution buffer, free of the contaminating proteins or proteinimpurities that were earlier allowed to pass through the column. Anycomponent can be used as a ligand for purifying its respective specificbinding protein, e.g. antibody. However, in various methods according tothe present invention, Protein A is used as a ligand for an Fc regioncontaining target protein or an antibody. The conditions for elutionfrom the ligand (e.g., Protein A) of the target protein (e.g., an Fcregion containing protein) can be readily determined by one of ordinaryskill in the art. In some embodiments, Protein G or a functional variantmay be used as a ligand. In some embodiments, a ligand such as Protein Ais used at a pH range of 5-9 for binding to an Fc region containingprotein, washing or re-equilibrating the ligand/target proteinconjugate, followed by elution with a buffer having pH about or below 4.

Although, affinity chromatography is specific for binding the protein ofinterest, affinity chromatography employing use of ligands such asProtein A and Protein G tends to be quite expensive and rapid fouling ofthe chromatography columns by non-specific materials (e.g., one or moreimpurities) poses a huge problem in the industry. The methods accordingto the present invention provide a solution to this problem by use ofmaterials (e.g., activated carbon) which reduce the burden ofchromatography columns by removing one or more of such non-specificmaterials from the sample, thereby decreasing the overall cost as wellas increasing the lifespan of the columns. Further, some of the methodsaccording to the claimed invention result in the use of fewerchromatography steps following the affinity chromatography step, therebyincreasing the efficiency of the overall process.

In a multi-step purification of recombinantly-produced proteins, it isusually beneficial to isolate the target protein from a diverse array ofsoluble impurities present in the cell culture fluid early in theprocess. This isolation can be achieved either by chromatographiccapture or by non-chromatographic isolation of target protein.

A chromatographic “capture” step, as used herein, consists of bindingtarget protein to a chromatography media positioned just downstream ofthe harvested feedstock produced either by a bacterial fermentation orby cell culture expression. Typically, the harvested feedstock isclarified, however capture can be accomplished from unclarifiedfeedstock as well. The primary function of this step is to bind thetarget protein from solution using the smallest amount of resinpossible, while allowing the impurities to flow through. The targetprotein is then eluted into a significantly smaller volume of buffer forfurther downstream processing. The chromatography media is selectedwhich has the best combination of dynamic binding capacity, massrecovery, and retention of the target's biological activity. Forantibodies containing Fc binding region, the use of an affinitychromatography media, such as those based on Protein A or Protein G, iscommon.

The chromatography media used for capture is chosen from the groupcomprising porous resin, membrane, monolith, woven or non-woven porousmaterials.

Non-chromatographic isolation of the target protein can be accomplishedby one or more of the following steps: protein adsorption on a solidporous or non-porous support; protein adsorption at a liquid-air orliquid-gas interface; protein adsorption at the interface between twoimmiscible or partially miscible liquids; protein precipitation as apure component or as a result of complex formation with one or moreother molecules or polymers; or by using protein crystallization.

The term “ion-exchange” and “ion-exchange chromatography” refers to thechromatographic process in which a solute or analyte of interest (e.g.,an Fc region containing target protein) in a mixture interacts with acharged compound linked by, e.g., covalent attachment, to a solid phaseion exchange material such that the solute or analyte of interestinteracts non-specifically with the charged compound more or less thansolute impurities or contaminants in the mixture. The contaminatingsolutes in the mixture elute from a column of the ion exchange materialfaster or slower than the solute of interest or are bound to or excludedfrom the resin relative to the solute of interest. “Ion-exchangechromatography” specifically includes cation exchange, anion exchange,and mixed mode ion exchange chromatography. For example, cation exchangechromatography can bind the target molecule (e.g., an Fc regioncontaining target protein) followed by elution (cation exchange bind andelution chromatography or “CIEX”) or can predominately bind theimpurities while the target molecule “flows through” the column (cationexchange flow through chromatography FT-CIEX). Anion exchangechromatography can bind the target molecule (e.g., an Fc regioncontaining target protein) followed by elution or can predominately bindthe impurities while the target molecule “flows through” the column. Insome embodiments and as demonstrated in the Examples set forth herein,the anion exchange chromatography step is performed in a flow throughmode. In a particular embodiment, the anion exchange chromatography stepemploys the use of a porous sorptive media comprising a porous substrateand a porous coating on the substrate, where the coating comprising oneor more polymeric primary amines or copolymers thereof.

The term “mixed-mode chromatography” or “multi-modal chromatography,” asused herein, refers to a process employing a chromatography stationaryphase that carries at least two distinct types of functional groups,each capable of interacting with a molecule of interest. An example ofmixed mode chromatography media is Capto™ Adhere (GE Healthcare), whichis an AEX mixed mode resin. Mixed-mode chromatography generally employsa ligand with more than one mode of interaction with a target proteinand/or impurities. The ligand typically includes at least two differentbut co-operative sites which interact with the substance to be bound.For example, one of these sites may have a charge-charge typeinteraction with the substance of interest, whereas the other site mayhave an electron acceptor-donor type interaction and/or hydrophobicand/or hydrophilic interactions with the substance of interest. Electrondonor-acceptor interaction types include hydrogen-bonding, π-π,cation-π, charge transfer, dipole-dipole and induced dipoleinteractions. Generally, based on the differences of the sum ofinteractions, a target protein and one or more impurities may beseparated under a range of conditions.

The term “hydrophobic interaction chromatography” or “HIC,” as usedherein, refers to a process for separating molecules based on theirhydrophobicity, i.e., their ability to adsorb to hydrophobic surfacesfrom aqueous solutions. HIC is usually differentiated from the ReversePhase (RP) chromatography by specially designed HIC resins thattypically have a lower hydrophobicity, or density of hydrophobic ligandscompared to RP resins.

HIC chromatography typically relies on the differences in hydrophobicgroups on the surface of solute molecules. These hydrophobic groups tendto bind to hydrophobic groups on the surface of an insoluble matrix.Because HIC employs a more polar, less denaturing environment thanreversed phase liquid chromatography, it is becoming increasing popularfor protein purification, often in combination with ion exchange or gelfiltration chromatography.

The terms “ion exchange resin,” “ion exchange media,” and “ion exchangematerial” refer to a solid phase that is negatively charged (i.e. acation exchange resin) or positively charged (i.e. an anion exchangeresin). The charge may be provided by attaching one or more chargedligands to the solid phase, e.g. by covalent linking or non-covalentcoating or adsorption. Alternatively, or in addition, the charge may bean inherent property of the solid phase.

The terms “CEX,” “cation exchange media,” “cation exchange resin” and“cation exchange material,” as used herein, refer to a solid phase whichis negatively charged, and which thus has free cations for exchange withcations in an aqueous solution passed over or through the solid phase. Anegatively charged ligand attached to the solid phase to form the cationexchange resin may, e.g., be a carboxylate or sulfonate. Commerciallyavailable cation exchange resins include carboxy-methyl-cellulose,sulphopropyl (SP) immobilized on agarose (e.g., SP-SEPHAROSE FAST FLOW™or SP-SEPHAROSE HIGH PERFORMANCE™, from Pharmacia) and sulphonylimmobilized on agarose (e.g. S-SEPHAROSE FAST FLOW™ from Pharmacia).

The terms “mixed mode media,” “mixed mode resin” and “mixed mode ionexchange resin,” as used herein, refer to a solid phase which iscovalently modified with cationic, anionic, and hydrophobic moieties. Acommercially available mixed mode ion exchange resin is BAKERBOND ABX™(J. T. Baker, Phillipsburg, N.J.) containing weak cation exchangegroups, a low concentration of anion exchange groups, and hydrophobicligands attached to a silica gel solid phase support matrix.

The term “HIC media” or “HIC resin” or “HIC material,” as used herein,refers to a chromatography material used for HIC separation. HIC mediais usually derived from porous chromatography resin modified withhydrophobic ligands, such as short aliphatic or aromatic groups.Examples of HIC media include Butyl Sepharose FF and Phenyl SepharoseFF, both commercially available from GE Healthcare. Additional examplesof commercial HIC resins include Fractogel® Phenyl and Fractogel® Propyl(MERCK KGA, Darmstadt, Germany), Butyl Sepharose® and Phenyl Sepharose®(GE HEALTHCARE).

The terms “AEX,” “anion exchange media,” “anion exchange resin” and“anion exchange material,” as used herein, refer to a solid phase whichis positively charged, e.g. having one or more positively chargedligands, such as primary, secondary, tertiary, or quaternary aminogroups, attached thereto. Commercially available anion exchange resinsinclude DEAE cellulose, QAE SEPHADEX™ and FAST Q SEPHAROSE™ (PHARMACIA).

The terms “Protein A” and “ProA” are used interchangeably herein andencompasses Protein A recovered from a native source thereof, Protein Aproduced synthetically (e.g., by peptide synthesis or by recombinanttechniques), and variants thereof which retain the ability to bindproteins which have a CH₂/CH₃ region, such as an Fc region. Protein Acan be purchased commercially from Repligen, GE Healthcare and Lonza.Protein A is generally immobilized on a solid phase support material.The term “ProA” also refers to an affinity chromatography resin orcolumn containing chromatographic solid support matrix to which iscovalently attached Protein A.

A functional derivative, fragment or variant of Protein A used in themethods according to the present invention may be characterized by abinding constant of at least K=10⁻⁸ M, and preferably K=10⁻⁹ M, for theFc region of mouse IgG2a or human IgG1. An interaction compliant withsuch value for the binding constant is termed “high affinity binding” inthe present context. Preferably, such functional derivative or variantof Protein A comprises at least part of a functional IgG binding domainof wild-type Protein A, selected from the natural domains E, D, A, B, Cor engineered mutants thereof which have retained IgG bindingfunctionality.

A “contaminant Protein A” according to the present invention is any typeof functional, IgG binding offspring of a Protein A or a functionalderivative thereof as defined above which is obtained upon eluting boundantibody from a Protein A affinity chromatography column. Suchcontaminant Protein A species may result e.g. from hydrolysis of peptidebonds which is very likely to occur by means of enzyme action inparticular in industrial manufacturing. Protein A chromatography isapplied as an early step in downstream processing when the crudelypurified, fresh product solution still harbors considerable proteaseactivity. Dying cells in the cell culture broth or cells disrupted ininitial centrifugation or filtration steps are likely to have set freeproteases; for regulatory purposes, supplementation of the cell culturebroth with protease inhibitors prior or in the course of downstreamprocessing is usually not accomplished, in contrast to biochemicalresearch practice. Examples are Phenyl-methyl-sulfonyl-chloride (PMSF)or e-caproic acid. Such chemical agents are undesirable as additives inthe production of biopharmaceuticals. It is further possible thatrecombinant functional derivatives or fragments of Protein A are lessprotease resistant than wild-type Protein A, depending on the tertiarystructure of the protein fold. Amino acid segments linking individualIgG binding domains might be exposed once the total number of bindingdomains is reduced. Interdomain contacts may possible contribute to thestability of domain folding. It might also be that binding of antibodyby Protein A or said functional derivatives thereof influences orfacilitates susceptibility to protease action, due to conformationalchanges induced upon binding of the antibody.

“Binding” a molecule to a chromatography resin is meant exposing themolecule to chromatography resin under appropriate conditions(pH/conductivity) such that the molecule is reversibly immobilized in oron the chromatography resin by virtue of ligand—protein interactions.Non-limiting examples include ionic interactions between the moleculeand a charged group or charged groups of the ion exchange material and abiospecific interaction between Protein A and an immunoglobulin.

The term “wash buffer” or “equilibration buffer” are usedinterchangeably herein, refers to a buffer used to wash orre-equilibrate the chromatography resin prior to eluting the polypeptidemolecule of interest. In some cases, the wash buffer and loading buffermay be the same. “Washing” a chromatography media is meant to encompasspassing an appropriate buffer through or over the media.

An “elution buffer” is used to elute the target protein from the solidphase. The conductivity and/or pH of the elution buffer is/are usuallysuch that the target protein is eluted from the chromatography resin.

To “elute” a molecule (e.g., a polypeptide of interest or an impurity)from chromatography resin is meant to remove the molecule therefrom byaltering the solution conditions such that buffer competes with themolecule of interest for binding to the chromatography resin. Anon-limiting example is to elute a molecule from an ion exchange resinby altering the ionic strength of the buffer surrounding the ionexchange material such that the buffer competes with the molecule forthe charged sites on the ion exchange material.

The term “eluate,” as used herein, refers to a solution containing amolecule of interest obtained via elution as well as the flow-throughfraction containing target protein of interest obtained as a result offlow-through purification. In some embodiments, the term “eluate” refersto the elution pool from a bind and elute chromatography step (e.g., aProtein A affinity chromatography step). In some embodiments, the eluatefrom a Protein A affinity chromatography step flows into a flow-throughpurification process which employs activated carbon, with or without anintervening virus inactivation step.

The term “solid phase” or “porous substrate” or “base matrix” refers toa non-aqueous material to which one or more charged ligands can adhere.The solid phase may be a purification column, a discontinuous phase ofdiscrete particles that is porous or non-porous, a membrane, a woven ornon-woven fibers, a monolith or a filter etc. Examples of materials forforming the solid phase include polysaccharides (such as agarose andcellulose); and other mechanically stable matrices such as silica (e.g.controlled pore glass), poly(styrenedivinyl)benzene, polyacrylamide,polyvinyl ether, nylon, high molecular weight polyethylene (HDPE),polyethersulfone, ceramic, and derivatives of any of the above.

A “buffer” is a solution that resists changes in pH by the action of itsacid-base conjugate components. Various buffers which can be employeddepending, for example, on the desired pH of the buffer are described inBuffers. A Guide for the Preparation and Use of Buffers in BiologicalSystems, Gueffroy, D., ed. Calbiochem Corporation (1975). In some stepsof the methods of the claimed invention, a buffer has a pH in the rangefrom 2.0 to 4.0, or from 2.8 to 3.8. In other steps of the claimedinvention, a buffer has a pH in the range of 5.0 to 9.0. In other stepsof the claimed invention, a buffer has a pH in the range of 4.0 to 6.5.In yet other steps of the methods of the claimed invention, a buffer hasa pH lower than 4.0. Non-limiting examples of buffers that will controlthe pH in this range include MES, MOPS, MOPSO, Tris, HEPES, phosphate,acetate, citrate, succinate, and ammonium buffers, as well ascombinations of these.

The term “conductivity” refers to the ability of an aqueous solution toconduct an electric current between two electrodes. In solution, thecurrent flows by ion transport. Therefore, with an increasing amount ofions present in the aqueous solution, the solution will have a higherconductivity. The unit of measurement for conductivity is milliSiemensper centimeter (mS/cm or mS), and can be measured using a commerciallyavailable conductivity meter (e.g., sold by Orion). The conductivity ofa solution may be altered by changing the concentration of ions therein.For example, the concentration of a buffering agent and/or concentrationof a salt (e.g. NaCl or KCl) in the solution may be altered in order toachieve the desired conductivity. Preferably, the salt concentration ofthe various buffers is modified to achieve the desired conductivity asin the Examples below.

The “pI” or “isoelectric point” of a polypeptide refer to the pH atwhich the polypeptide's positive charge balances its negative charge. pIcan be calculated from the net charge of the amino acid residues orsialic acid residues of attached carbohydrates of the polypeptide or canbe determined by isoelectric focusing.

As used herein, “filtrate” refers to that portion of a sample thatpasses through a media.

As used herein, “retentate” refers to that portion of a sample that issubstantially retained by the media.

The term “clarification” as used herein, refers to a process forreducing turbidity, as measured in NTU, of a protein-containingsolution, by removing suspended particles. Clarification can be achievedby a variety of means, including batch and continuous centrifugation,depth filtration, normal and tangential flow filtration, andprecipitation, including flocculation with small molecule and polymericspecies, or any combinations of methods thereof.

The phrase “reducing the burden of chromatography column” refers to atreatment or processing of a protein containing sample, which treatmentor processing results in decreasing the fouling of a chromatographycolumn onto which the protein sample is subsequently loaded. In atypical chromatography experiment, there could be non-eluting substancesor impurities left behind on the stationary phase, thereby resulting infouling of a chromatography column. This problem is further intensifiedduring scale up, where the degree of column fouling is greater due togreater level of impurities being present. Fouling of columns alsoresults in significantly diminishing the life span of the columns, whichare usually quite expensive. The methods according to the presentinvention provide improved methods for significantly reducing the burdenof chromatography columns, thereby increasing the life span of thecolumns as well resulting in greater yield of protein. The methodsaccording to the present invention result in removal of significantamounts of impurities before loading the sample onto a chromatographycolumn, thereby reducing the burden of the column. Typical impuritiesinclude host cell protein, DNA, fatty acid (from cell debris), dyemolecule, defoaming agent, etc.

The term “process step” or “unit operation,” as used interchangeablyherein, refers to the use of one or more methods or devices to achieve acertain result in a purification process. Examples of process steps orunit operations which may be employed in the processes and systemsdescribed herein include, but are not limited to clarification, bind andelute chromatography capture, virus inactivation, flow-throughpurification and formulation. It is understood that each of the processsteps or unit operations may employ more than one step or method ordevice to achieve the intended result of that process step or unitoperation. For example, in some embodiments, the flow-throughpurification step, as described herein, may employ more than one step ormethod or device to achieve that process step or unit operation, whereat least one such step involves activated carbon. In some embodiments,one or more devices which are used to perform a process step or unitoperation are single use or disposable and can be removed and/orreplaced without having to replace any other devices in the process oreven having to stop a process run.

As used herein, the term “pool tank” refers to any container, vessel,reservoir, tank or bag, which is generally used between process stepsand has a size/volume to enable collection of the entire volume ofoutput from a process step. Pool tanks may be used for holding orstoring or manipulating solution conditions of the entire volume ofoutput from a process step. In some embodiments, the processes anddescribed herein obviate the need to use one or more pool tanks.

The term “surge tank” as used herein refers to any container or vesselor bag, which is used between process steps or within a process step(e.g., when a single process step comprises more than one step); wherethe output from one step flows through the surge tank onto the nextstep. Accordingly, a surge tank is different from a pool tank, in thatit is not intended to hold or collect the entire volume of output from astep; but instead enables continuous flow of output from one step to thenext. In some embodiments, the volume of a surge tank used between twoprocess steps or within a process step in a process or system describedherein, is no more than 25% of the entire volume of the output from theprocess step. In another embodiment, the volume of a surge tank is nomore than 10% of the entire volume of the output from a process step. Insome other embodiments, the volume of a surge tank is less than 35%, orless than 30%, or less than 25%, or less than 20%, or less than 15%, orless than 10% of the entire volume of a cell culture in a bioreactor,which constitutes the starting material from which a target molecule isto be purified. In some embodiments, a surge tank is employed during aflow-through purification process step which uses activated carbonfollowed by AEX chromatography followed by CEX chromatography followedby virus filtration, where the surge tank is used to perform solutionchange after the AEX chromatography step.

The term “continuous process,” as used herein, refers to a process forpurifying a target molecule, which includes two or more process steps(or unit operations), such that the output from one process step flowsdirectly into the next process step in the process, withoutinterruption, and where two or more process steps can be performedconcurrently for at least a portion of their duration. In other words, acontinuous process obviates the need for completing a process stepbefore performing the next process step in the purification process. Theterm “continuous process” also applies to steps within a process step,in which case, during the performance of a process step includingmultiple steps, the sample flows continuously through the various stepsthat are necessary to perform the process step. Accordingly, in someembodiments, a flow-through purification step employing activated carbonis performed in a continuous manner, where the eluate from a bind andelute chromatography step (e.g., Protein A chromatography capture step),which precedes the flow-through purification step, flows into anactivated carbon (packed in cellulose media) step followed by an AEXchromatography step followed by a CEX chromatography step and followedby a virus filtration step.

The term “static mixer” refers to a device for mixing two fluidmaterials, typically liquids. The device generally consists of mixerelements (also referred to as non-moving elements) contained in acylindrical (tube) housing. As the streams move through the staticmixer, the non-moving elements continuously blend the materials.Complete mixing depends on many variables including the properties ofthe fluids, inner diameter of the tube, number of mixer elements andtheir design etc. In some embodiments described herein, one or morestatic mixers are used in the processes described herein, e.g., betweenan AEX chromatography step and a CEX chromatography step.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures, are incorporated herein byreference.

II. Exemplary Carbonaceous Materials for Use in the Claimed Methods

In methods according to the present invention, certain carbonaceousmaterials such as, activated carbon, are used in the purification ofproteins. Activated carbon can be described as a porous solid with avery high surface area. In some embodiments, activated carbon comprisesactivated charcoal. Activated carbon can be derived from a variety ofsources including, but not limited to, coal, wood, coconut husk,nutshells, and peat. Activated carbon can be produced from thesematerials by physical activation involving heat under a controlledatmosphere or by chemical activation using strong acids, bases, oroxidants. The activation processes produce a porous structure with ahigh surface area that gives activated carbon a greater capacity forimpurity removal. Activation processes can be modified to control theacidity of the surface.

Activated carbon is available from a wide variety of commercial sourcesand comes in a number of grades and formats. Some of the commercialsuppliers of activated carbon include companies such as MeadWestVacoCorp., Richmond, Va., USA; Norit Americas Inc., Marshall, Tex., USA;Calgon Carbon Corp., Pittsburgh, Pa., USA.

Two major formats of activated carbon are powdered and granular.Powdered activated carbon contains small and usually less than 1 mmdiameter particles, and is most commonly used for purification ofliquids. Granular activated carbon has a larger particle size andconsequently a smaller surface area, so it is preferred for use in gaspurification where the rate of diffusion is faster.

An important consideration for safety with use of activated carbon inconsumer applications (such as water, food, beverage, and pharmaceuticalpurification) is reduction and control of extractable compounds.Activated carbon intended for drinking water and food contactapplications is usually made in compliance with safety standard ANSI/NSFStandard 61 that covers all indirect additives to water. Also, ASTMstandard test method D6385 describes determining acid extractablecontent in activated carbon by ashing and could be used to study andminimize the level of extractables from activated carbon.

A range of activated carbon types is available for various applications.For example, MeadWestVaco Corp. supplies at least twelve types ofpowdered activated carbon that vary by their capacity, surface acidity,pore accessibility to target molecules, and intended application. It isgenerally desirable to maximize the capacity of activated carbon forimpurity removal.

In some embodiments described herein, activated carbon is incorporatedin a cellulose media.

III. Conventional Flow-Through Purification Processes

Most conventional flow-through purification processes rely distinctly ondifferent surface interactions between the target protein of interestand the impurity to be removed. For example, conventional AEXflow-through purification of monoclonal antibodies relies on the factthat isoelectric point of most antibodies is higher than other proteinsand nucleic acids and is usually above 7. Thus, AEX flow-throughpurification processes are carried out at a pH that is lower than the pIof the antibody being purified in order to make sure that the stationaryphase and the antibody have the same charge and thus the antibody flowsthrough the media without significantly binding to the surface. On theother hand, many proteinaceous impurities nucleic acids, and endotoxinshave a pI lower than an antibody, which is usually below 7, andaccordingly, they bind to the surface of an AEX media.

Like most ion-exchange chromatography processes, AEX flow-throughpurification is generally sensitive to solution conductivity and isgenerally less effective at higher salinity. In a typical purificationprocess of a monoclonal antibody, AEX flow-through purification,sometimes referred to as the “polishing step,” follows one or more bindand elute column chromatography steps.

Two most commonly used process templates are shown below:

1) Protein A capture→CEX bind and elute purification andconcentration→Dilution→AEX flow-through

-   -   2) Protein A capture→AEX flow-through→CEX bind and elute        purification and concentration

In addition to these commonly employed process templates, otherpurification schemes are sometimes employed, including CEX capture andthe use of mixed-mode and inorganic bind and elute resins (such asCeramic Hydroxyapatite, CHT). In general, the goal of flow-throughpurification is to remove trace levels of impurities, whereas the bulkof purification is done using bind and elute steps. However, a shiftfrom using primarily bind and elute steps to a flow-through purificationprocess can be a very cost-effective solution that saves time, reagentsas well as operational costs. Accordingly, the processes describedherein provide a viable solution to the conventional processes in thatthey are more cost-effective and reduce the overall manufacturing andoperational costs.

In some embodiments described herein, improved flow-through purificationprocesses are provided which enable flow-through purification to beperformed in a continuous manner.

IV. Use of Carbonaceous Material in Purification Processes

As discussed above, the present invention provides novel and improvedpurification processes which employ activated carbon. Activated carboncan be added directly to a purification step and can subsequently beremoved by sedimentation or filtration, or by passing solution or gasthrough a device containing activated carbon. Activated carbon caneither be packed independently into a suitable device or it may beblended with other materials that enhance its mechanical, flow, orseparation properties. For example, activated carbon can be incorporatedinto a wet-laid fibrous media containing cellulose, and then sealedinside a disposable device such as Millistak+® Pod CR available fromMillipore Corporation, or Seitz® AKS Filter Media available from PallCorporation, Port Washington, N.Y., USA. Another format of activatedcarbon is an activated carbon block, where activated carbon isincorporated into a porous monolith by pressing together withthermoplastic powder. Granular form of activated carbon can also bepacked into columns, similar to chromatography media, or it may bepacked into a suitable device. It is generally accepted that activatedcarbon media is used for decolorization, removal of small moleculeimpurities, etc. For example, there are several grades of activatedcarbon media available from Pall Corporation, that can be selected basedon molecular weight of targeted impurities. Three molecular weightranges are available: 200-400, 400-1,000, and 400-1,500 Daltons, thelatter being the largest. However, none of the commercially availableactivated carbon media have been described for the selective removal ofmuch larger impurities from biological samples, ranging from 2000 to200,000 Daltons in molecular weight.

The present invention is based, at least in part, on the surprisingdiscovery that activated carbon is capable of selectively bindingundesirable impurities (e.g., HCPs and DNA), while at the same timeshowing negligible binding to a target protein.

This invention also recognizes the fact that, similar to all adsorptivematerial, the adsorptive capacity of activated carbon is not unlimited.For example, in the case of CHO-S feed, activated carbon has shown toeffectively remove both HCP and DNA when these species were present atconcentrations that were observed in representative feeds as shown inExample 1. However, addition of DNA to an unusually high level was foundto suppress the HCP removal efficiency of activated carbon as shown inthe Example 2.

Activated carbon can also be highly beneficial in removing potentialcomponents of cell culture media that may be present in the solution oftarget protein, both before and after target protein capture step.Typical components of cell culture media include surfactants (e.g.,Pluronic® F68), insulin, antibiotics, methotrexate, and antifoam. Due toa risk that some of these components will be carried over into apurified target protein, it is advantageous to incorporate a step into aprotein purification train that is capable of removing these components.Activated carbon may be used in a purification process to remove suchcomponents, as further evidenced by Examples herein.

The following are examples of protein purification processes thatincorporate activated carbon as one or more intermediate steps, which isshown by underline in the following Table I below. It is understood thatmany variations of these processes can be used.

TABLE I Step 1 Step 2 Step 3 Step 4 Step 5 Process A Provide Flow Bindand Bind and Flow Clarified through elute with elute with through CellCulture activated Affinity CEX AEX Fluid carbon media media mediaProcess B Provide Bind and Flow Bind and Flow Clarified elute withthrough elute with through Cell Culture Affinity activated CEX AEX Fluidmedia carbon media media Process C Provide Bind and Bind and Flow FlowClarified elute with elute with through through Cell Culture AffinityCEX activated AEX Fluid media media carbon media Process D Provide Bindand Bind and Flow Flow Clarified elute with elute with through throughCell Culture Affinity CEX AEX activated Fluid media media media carbonProcess E Provide Bind and Flow Flow Clarified elute with throughthrough Cell Culture Affinity activated AEX Fluid media carbon mediaProcess F Provide Bind and Flow Flow Clarified elute with throughthrough Cell Culture CEX activated AEX Fluid media carbon media ProcessG Provide Bind and Flow Flow Flow Clarified Elute with through throughthrough Cell Culture Affinity activated AEX CEX Fluid media carbon mediamedia

In general, in the Table above, the step of Bind and Elute with Affinitymedia, and/or Bind and Elute with CEX media can be operated in any ofthree modes: (1) batch mode, where the media is loaded with targetprotein, loading is stopped, media is washed and eluted, and the pool iscollected; (2) semi-continuous mode, where the loading is performedcontinuously, while the elution is intermittent (e.g., in case ofcontinuous multicolumn chromatography); and (3) full continuous mode,where both loading and elution are performed continuously.

In some embodiments, one or more processes described in the Table above,a virus inactivation step may be performed after the bind and elute stepand before subjecting the eluate to a flow-through purification step, asdescribed herein.

It is understood that the processes described herein and in the Tableabove may further employ additional steps as well as steps for changingsolution conditions in-line or via a surge tank. In some embodimentsdescribed herein, a process includes the following steps: clarification;bind and elute with Protein A affinity media; in-line virusinactivation; flow-through purification as follows: activated carbonfollowed by flow-through AEX media followed by a solution change usingan in-line static mixer and/or surge tank, followed by flow-through CEXmedia followed by virus filtration; and formulation.

V. Assaying for Reduced Levels of One or More Impurities

The present invention provides processes for reducing the level of oneor more impurities present in a sample containing a protein of interest.Typical impurities contained in a protein sample derived from abiological source include host cell proteins (HCP) and nucleic acids(DNA). When a host cell is Chinese Hamster Ovary (CHO), the HCP iscommonly referred to as CHO HCP or CHOP. Immunological methods usingantibodies to HCPs such as Western Blot and ELISA are conventionallyused for detection of such impurities. Microtiter plate immunoenzymetricassays (ELISA) are also routinely employed in order to provide a highsensitivity of analysis. Such assays are simple to use, objective, andpowerful tools for measuring the level of one or more impurities duringpurification processes.

Some of the ELISA kits for measuring HCP are commercially available fromvendors such as, e.g., Cygnus Technologies of Southport, N.C., USA. Someof such kits are “generic” in the sense that they are intended to reactwith essentially all of the HCPs that could contaminate the productindependent of the purification process that was used. In someembodiments, commercially available kits may be used for detecting thelevel of one or more impurities in a sample.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures, are incorporated herein byreference.

EXAMPLES Example 1. Evaluation of Various Materials for Impurity Removalin Flow-Through Mode

In this experiment, different materials were evaluated for their abilityto remove impurities from a clarified CHO feed. Materials, shown inTable II, were tested for impurity removal in flow-through mode.Phosphate buffer saline (PBS, 10 mM phosphate, pH 7.4) was used as anequilibrium and wash buffer.

TABLE II Material Acronym Description Vendor/Catalog Activated AC RGC 80MeadWestVado carbon SP sepharose SP FF Agarose cation GE HealthcareFastFlow exchange chromatography (CIEX) resin ProRes ™-S ProRes ™-SPolymeric cation Millipore exchange Corporation chromatography (CIEX)resin Q sepharose Q FF Agarose anion GE Healthcare FastFlow exchangechromatography (AIEX) resin Phenyl Ph FF Agarose hydrophobic GEHealthcare Sepharose interaction FastFlow chromatography(HIC) resin

Gravity flow-through (FT) test method was used to test resin impurityremoval. One ml of each material listed in Table II (1 ml) was allowedto settle and packed into a 5 ml disposable chromatography column(Evergreen Scientific, LA, Calif.). The columns were equilibrated with 5column volumes (CVs) of equilibrium buffer (PBS), loaded with 20 CVs offeed and washed with 5 CVs of wash buffer (PBS) again. The flow-througheluant fractions were collected. Untreated clarified null CHO-S feedwith the addition of a polyclonal IgG (˜2.6 mg/ml, SeraCare) was used.

Flow-through eluant and the corresponding feed were tested for IgGyield, UV active species (280 nm) removal, HCP removal and DNA removal.IgG yield was quantified on a Waters Alliance HPLC (Milford, Mass.)using Poros Protein A 2.1 mm×30 mm analytical column (LIFE TECHNOLOGIES,Palo Alto, Calif.) per vendor's instructions. UV active species at 280nm was quantified using peak area of the non-retaining species that areshown as the flow through peak on the Protein A chromatogram. Host cellprotein was detected using the CHO-CM HCP ELISA kit (CYGNUSTECHNOLOGIES, Southport, N.C.). DNA was detected using Quant-iT™PicoGreen® dsDNA Reagent (LIFE TECHNOLOGIES, Foster City, Calif.). Allassays were done according to manufactures protocols. The results aredepicted in FIGS. 1-4 .

As depicted in FIG. 1 , except for HIC resin, which shows ˜5% IgG yieldloss, all other media screened, i.e., activated carbon, SPFF, ProRes™-S,and QFF demonstrated no detectable loss of yield.

Further, as depicted in FIGS. 2, 3 and 4 , activated carbon resulted ina significant reduction in the levels of UV active species at 280 nm.This includes pH indicators, some population of HCP, DNA and someresidual cell culture components. In the case where polyclonal IgG wasused, this fraction also includes the IgG 3 population that Protein Adoes not bind. While all materials appeared to remove HCP to somedegree, including activated carbon, the two materials with cationexchange functionality, Sepharose FastFlow and ProRes™-S, removed themost HCP. As for DNA removal, while it was not surprising to seesignificant DNA removal by anion exchange resin, Q FastFlow, it wasunexpected to see that activated carbon removed DNA in similar capacity.All other material removed DNA to a lesser degree.

Data from this example demonstrates that activated carbon, as well asmaterials with cation exchange, anion exchange and hydrophobicinteraction functional groups, can remove impurities (HCP, DNA and UVactive species) to various degrees without significant yield loss inflow through mode from a CHO feed.

Example 2. Flow-Through Impurity Removal Capacity

In a representative experiment, the amount of impurities removed bydifferent materials per unit volume was evaluated. The level of DNA inthe CHO-S feed was increased through the addition of commerciallyavailable Herring Sperm DNA to understand the effect of adsorptioncompetition between DNA and HCP. The materials that were evaluated forimpurity removal are listed in Table III below.

TABLE III Material Acronym Description Vendor/Catlog Activated ACactivated carbon MeadWestVaco carbon RGC 80 SP sepharose SP FF cationexchange GE Healthcare, FastFlow chromatography Cat# 17-0729-01 (CIEX)resin, agarose base matrix ProRes ™-S ProRes ™-S cation exchangeMillipore chromatography Corporation (CIEX) resin, polymeric base matrixQ sepharose Q FF Anion exchange GE Healthcare, FastFlow chromatographyCat# 17-0510-01 (AIEX) resin, agarose base matrix

Each of the materials listed in Table III (1 ml) were packed into anOmnifit column (0.66 cm i.d.). Columns were equilibrated with 5 CVs PBS,loaded with 100 CV of null untreated clarified CHO-S feed with theaddition of polyclonal IgG and Herring sperm DNA and washed with 20column volumes PBS on a BioCad (APPLIED BIOSYSTEMS, Palo Alto, Calif.).The flow-through eluant fractions during loading step were collected atevery 10 column volumes. Flow-through eluant fractions and thecorresponding feeds were assayed for IgG yield, UV active species, HCPand DNA removal using the same methods as described in Example 1.

As depicted in FIG. 5 , all materials screened showed no significantloss of IgG yield over 100 column volumes loading of feed. As depictedin FIG. 6 , activated carbon removed the most UV active speciesthroughout the 100 CVs. Anion exchange material also removed UV activespecies to some degree throughout the 100 CVs. Further, as depicted inFIG. 7 , HCP removal capacity by anion exchange resin, Q FastFlow, waslimited as it breaks through from early fractions. However, cationexchange materials, Q FastFlow and ProRes™-S, removed a significantamount of HCP throughout the 100 column volumes. With the high DNAconcentration (i.e., an additional 195 pg/mL) in this specificexperiment, activated carbon removed less HCP compared to the feedswithout the added DNA. For example, in a similar experiment with theoriginal null CHO-S feed without the addition of DNA, activated carbonremoved close to 20% HCP throughout the 100 CVs (data not shown). Thisdemonstrated some degree of competition of DNA and HCP for theadsorption to activated carbon. Further, as depicted in FIG. 8 , allmaterials removed DNA to some extent.

Data from this example demonstrates that activated carbon, as well asmaterials with cation exchange, anion exchange and hydrophobicinteraction functional groups, can remove impurities (HCP, DNA and UVactive species) to various degrees over 100 column volumes, withoutsignificant yield loss in flow through mode from a CHO cell feed.

Example 3. Effect of Combinations of Different Materials on ImpurityRemoval

In another experiment, different combinations of materials, shown in thework flow depicted in FIG. 9 , were evaluated for impurity removal. Thematerials (1 ml) were allowed to settle and packed into a 5 mldisposable chromatography column. Columns were equilibrated with 5 CVsof PBS, loaded with 20 CVs feed and washed with 5 CVs of PBS.

The flow-through eluant fraction was further loaded on o the nextdisposable column of the selected media (as shown in the two workflowsdepicted on the left in FIG. 9 ). The last flow-through eluant in thework flow was analyzed. In case of the work flow depicted on the right,in which a feed went through a mixture of media, the eluant was analyzeddirectly. Feed was prepared using non-expressing CHO-S feed withaddition of polyclonal IgG (˜2.5 mg/ml) from SeraCare. Flow-througheluant fractions and the corresponding feeds were assayed for IgG yield,UV active species, HCP and DNA removal using the same methods describedabove in Example 1. Results are shown in FIGS. 10-12 .

As depicted in FIG. 10 , activated carbon and mixtures which containactivated carbon significantly reduced the level of UV active species.Further, while all materials exhibited HCP removal to some extent,activated carbon mixture in combination with a cationic resin showedmost effective removal of HCP, as depicted in FIG. 11 . Also, asdepicted in FIG. 12 , while all materials removed DNA to some extent,combination of activated carbon and anion exchange resin showed mosteffective removal of DNA.

Data from this example demonstrates that combination of activated carbonand materials with cation exchange, anion exchange and hydrophobicinteraction functional groups can remove impurities (HCP, DNA and UVactive species) more effectively than any single component in flowthrough mode from a CHO feed.

Example 4. Impurity Removal of a Protein A Elution Pool

In another experiment, standard gravity flow-through test method wasused to investigate impurity removal in a post Protein A pool. Materialsscreened in Example 1, i.e., activated carbon, cation exchange resins(SP FF and ProRes™-S), anion exchange resin, Q FF, and HIC resin, PhenylFastFlow and different combinations of these materials, were evaluated.Activated carbon or resin material (1 ml) was allowed to settle andpacked into a disposable chromatography column from Evergreen. Eachcolumn was equilibrated with 5 CVs of PBS, loaded with 20 CVs of feedand washed with 5 CVs of PBS. The flow-through eluant fractions duringloading step were collected. The feed used was Protein A (ProSep UltraPlus) elution pool (˜3.2 mg/ml IgG) after adjusting pH to 7.0. The feedfor Protein A column was a non-expressing CHO-S spiked with polyclonalIgG.

Flow-through eluant fractions and the Protein A elution pool wereevaluated for IgG yield, UV active species removal, HCP removal and DNAremoval. All assays were performed as described in Example 1, exceptthat in case of HCP, CHO-3G HCP ELISA kit (CYGNUS TECHNOLOGIES,Southport, N.C.) was used.

As depicted in FIG. 13 , all materials screened generated ˜85% or higheryield. Activated carbon, cation exchange resin, SP FastFlow, removed themost HCP as a single material. The mixture of different materialsprovided the highest HCP removal, as depicted in FIG. 14 . FIG. 15depicts the fact that all material removes significant amount of DNA,partly because of the low level in the feed. Overall, mixtures ofmaterials were generally more effective in impurity removal compared tosingle materials.

Data from this example demonstrates that activated carbon and materialswith cation exchange, anion exchange and hydrophobic interactionfunctional groups, as well as the mixture of these materials can removeimpurities (HCP, DNA and UV active species) to different degrees in flowthrough mode from a Protein A elution pool generated from a CHO feed.

Example 5. Preparation of a Representative Affinity Based (Protein A)Captured MAb Feed to Evaluate the Performance of Activated Carbon forthe Purification of MAb

A partially purified monoclonal antibody, referred to as MAb I, wasproduced using a CHO cell line culture for use as a representativeaffinity (Protein A) captured monoclonal antibody feed to evaluate theperformance of activated carbon for purifying proteins. The cell culturewas first clarified directly from a bioreactor using depth filtrationmedia, available as Millistak+® POD filters (MILLIPORE CORPORATION,Billerica, Mass., USA). Cell culture fluid was filtered through a seriesof two filters, DOHC and XOHC to a final turbidity of <10 NTU, andsubsequently sterile filtered with Millipore Express® SHC capsulefilter.

An acrylic Quick-Scale® 14 cm ID column from Millipore Corporation,Billerica, Mass., USA was packed with Millipore ProSep-vA High CapacityProtein A media to a bed volume of approximately 3.2 L. The column waspacked using a combination of flow packing with PBS and vibration. Allchromatography steps were performed on a Millipore K-Prime 40-I systemwith detection performed using UV absorption at a wavelength of 280 nm.The column was used for MAb I purification for at least five cycles andwas stored in PBS at 4° C. Prior to using the column, the column wasflushed with at least 2 column volumes (CVs) of 0.15 M phosphoric acidpH 1.5-1.7 and then equilibrated with PBS until the pH was stabilized(at least 3 CVs). Typically, the day following the clarification, thesterile filtered clarified cell culture was loaded onto the Protein Acolumn at a residence time of at least 5 minutes (i.e., at a flow rateof 500-600 mL min⁻¹). The column was loaded to ensure that the maximumcapacity of the column was not exceeded, which was defined as 30 g ofMAb I per liter of media.

Following loading of the column, the column was flushed with PBS at thesame residence time until the UV trace reached baseline, typicallywithin three CVs. The column was then washed with 20 mM sodium acetatewith 0.5 M NaCl at pH 6 for at least three CVs, but for no more thanfive CVs. The product was then eluted using a step change to 20 mMacetic acid pH 3.0, where the capture of the elution peak was performedmanually to reduce the dilution of the elution peak. The eluate was leftto incubate at room temperature (20-25° C.) for at least 30 minutes to amaximum of 1.5 hours. Following incubation, the pH of the elution poolwas titrated to pH 5±0.2 using 2 M Tris base at pH 10 or higher. Thestarting pH of the elution pool was typically near pH 4.0 and requiredless than 5% volume addition of the Tris base solution. During thetitration of the elution pool visible precipitates were observed. Theremoval of the precipitates was performed using a Millipore Millistak+0XOHC lab scale POD prior to sterile filtration of the elution pool,where at least two 0.027 m² PODs were required to clear the precipitatesof the entire elution pool. Following the depth filtration, the eluatewas sterile filtered using Millipore Express Plus Stericup filter unitsand stored at 4° C. until used.

Example 6. Preparation of a Representative Non-Affinity Based (CationExchange) Captured MAb Feed to Evaluate the Performance of ActivatedCarbon for the Purification of MAb

A partially purified monoclonal antibody, referred to as MAb I, wasproduced using a CHO cell line culture for use as a representativenon-affinity (cation exchange) captured monoclonal antibody feed toevaluate the performance of activated carbon for purifying proteins. AMAb I clarified cell culture fluid was made as discussed in Example 5.Cell culture fluid was filtered through a series of two filters, DOHCand XOHC to a final turbidity of <10 NTU, and subsequently sterilefiltered with Millipore Express® SHC capsule filter.

A 22 cm ID glass Vantage® laboratory column from Millipore Corporation,Billerica, Mass., USA was packed with the cation-exchange (CEX) mediaFractogel® SO₃ ⁻ (M). The column was packed using PBS on an Akta®Explorer 100 (GE HEALTHCARE, Uppsala, Sweden) with superficial linearvelocities of approximately 1000 cm hr⁻¹. The pressure drop across thecolumn was kept below three bar during packing and all subsequent columnruns. The bed compression of the column was approximately 15% with apacked bed volume of 75 mL. The packing efficiency of the column wasmeasured using a pulse injection of 5004 of 25 mM Tris, 1 M NaCl, 3%(v/v) acetone at pH 6.9 at a superficial linear velocity of 100 cm hr⁻¹using PBS as the running buffer. The height equivalent to a theoreticalplate (HETP) was calculated using the acetone peak using standardmethods after accounting for the dead volume of the system and wascalculated as 0.053 cm with an asymmetry of the peak of 1.3, indicatingthe column was packed sufficiently. The NaCl peak showed a larger levelof peak tailing, but this is likely related to the interaction of thesalt ions with the media and not a representation of the columnefficiency.

Prior to the first use of the column, a complete blank run was performedwithout a protein load so as to reduce and/or eliminate any solutionrelated interactions with the base media. Prior to loading, the columnwas equilibrated with 20 mM sodium acetate pH 5 for at least five CVs.The column was loaded with a clarified cell culture containing MAb Ithat had the pH lowered to pH 5 using glacial acetic acid. When loweringthe pH of the cell culture, precipitates were often observed and wereremoved using a combination of centrifugation and depth filtration usinga Millipore Millistak+0 XOHC lab scale POD.

The loading solution was sterile filtered prior to loading usingMillipore Express Plus Stericup filter units. The sterile filtered loadwas loaded onto the column using a residence time of 10 min in an effortto maximize the column capacity (approximately 45 g MAb I per L ofmedia), while minimizing the pressure drop across the column. Followingloading, the column was flushed with equilibration buffer for five CVs.The column was then washed with 20 mM sodium acetate, 0.1 M NaCl pH 5.0buffer for five CVs. The elution from the column was performed with astep change to 20 mM sodium acetate, 0.25 M NaCl pH 6.0 for at leastfive CVs. The eluate was fractionated and the peak fractions were pooledand stored at 4° C. for further use. The column was then washed with 20mM sodium acetate, 1 M NaCl pH 6.0 to remove strongly bound proteins,which were determined to be mostly host cell proteins (HCP). The columnwas then washed with 0.5 N NaOH for five CVs at a slower flow rateequivalent to a 30 minute residence time. The column was then washedwith five CVs of the equilibration buffer and stored at room temperature(20-25° C.) for future use.

Example 7. Evaluation of Various Adsorbers by Flow-Through Treatment ofan Affinity Captured MAb Eluate

Activated carbon was compared in a flow-through application to severaldifferent commercially available adsorbent media that are commonly usedfor the purification of proteins including anion exchange (ChromaSorb™),cation exchange (HiTrap SP FF, HiTrap CM FF), and hydrophobicinteraction (HiTrap Phenyl FF, HiTrap Butyl FF) chemistries for thepurification of an affinity (Protein A) captured monoclonal antibodyeluate to demonstrate that the activated carbon is unique andunexpectedly efficient for the removal of impurities from proteinsolutions.

A partially purified MAb I affinity (Protein A) captured eluate wasprepared according to Example 5. The eluate of MAb I was adjusted fromapproximately pH 5 to pH 7 with Tris base (2 M) and filtered through a0.22 micron Millipore Express Plus Stericup filter unit. The solution isreferred to herein as the MAb I Protein A eluate.

Nuchar HD and HD Nuchar activated carbon are used herein interchangeablyand refer to the grade of powdered activated carbon obtained fromMeadWestVaco Corporation, Richmond, Va., USA. Glass OmnifitChromatography Columns (10 mm diameter, 100 mm length) were loaded with250 mg of HD Nuchar activated carbon slurried in water to give a packedcolumn volume of 1 mL. 0.2 mL ChromaSorb membrane devices weremanufactured using 0.65 micron-rated polyethylene membrane modified withpolyallyl amine, available from Millipore Corporation, Billerica, Mass.,USA, in devices of various sizes. The membrane was cut in 25 mm discs; 5discs were stacked and sealed in an overmolded polypropylene device ofthe same type as the OptiScale 25 disposable capsule filter devicescommercially available from Millipore Corporation. The devices includean air vent to prevent air locking, and have an effective filtrationarea of 3.5 cm² and volume of 0.2 mL.

1 mL pre-packed chromatography columns HiTrap SP FF, HiTrap CM FF,HiTrap Phenyl FF (high sub), and HiTrap Butyl FF were purchased from GEHealthcare, Pittsburgh, Pa., USA, and equilibrated with buffer solution(Tris-HCl buffer, 25 mM, pH 7) prior to use. Six flow-throughpurification trains were assembled: a) activated carbon, b) ChromaSorb,c) HiTrap SP FF, d) HiTrap CM FF, e) HiTrap Phenyl FF, and f) HiTrapButyl FF. Subsequently, 96 mL of the MAb I Protein A eluate was passedthrough each setup at a flow rate of 0.25 mL/min. After passing throughthe purification trains, the solutions were analyzed for host cellprotein (HCP), IgG concentration, and residual Protein A. HCP analysiswas performed using a commercially available ELISA kit from CygnusTechnologies, Southport, N.C., USA, catalog number F550, following kitmanufacturer's protocol. IgG concentration was measured using an AgilentHPLC system equipped with a Poros® A Protein A analytical column.Protein A analysis was performed using a commercially available ELISAkit from Meridian Life Sciences, Saco, ME, USA, Kit COZ51-188. Resultsare summarized in Table IV.

The results show that activated carbon removed the greatest amount ofimpurities with a log reduction value (LRV) of host cell protein (HCP)of 0.87. The LRV of HCP value for activated carbon was significantlyhigher in comparison to the commercially available media (anionexchange, cation exchange, hydrophobic interaction) examined, which hadLRV of HCP that ranged from 0.19 to 0.35. The residual Protein A fromaffinity capture step was also efficiently removed below the detectablelimit by activated carbon. The only commercially available media thatwas observed to remove any significant amount of residual Protein A wasthe anion exchange media (ChromaSorb™), which also lowered theconcentration of residual Protein A below the detectable limit. Despitethe higher amounts of impurities removed by the activated carbon itstill had excellent recovery (96%) of the monoclonal antibody product(MAb I), which was similar to the product recoveries (82-100%) observedfor the commercially available media that were examined.

TABLE IV flow-through MAb I Recovery HCP HCP LRV of Protein A Protein Aadsorber (mg/mL) MAb I (ng/mL) (ppm) HCP (ng/mL) (ppm) Untreated(control) 7.18 NA 869 121 NA 4.61 0.62 Activated carbon 6.88 96% 112 160.87 ND ND ChromaSorb 5.92 82% 320 54 0.35 ND ND HiTrap SP FF 6.09 85%364 60 0.31 2.95 0.48 HiTrap CM FF 6.28 87% 463 74 0.22 2.72 0.43 HiTrapPhenyl FF 6.89 96% 385 56 0.33 3.21 0.47 HiTrap Butyl FF 7.21 100%  56378 0.19 4.25 0.59 ND—Not Detected NA—Not Applicable

Example 8. Static Soak Treatment of an Affinity Captured Eluate of a MAbwith Activated Carbon and/or Anion Exchange Media

Activated carbon alone and in combination with an anion exchange mediawas examined for the removal of impurities from an affinity (Protein A)captured monoclonal antibody eluate to demonstrate a unique andunexpectedly effective method for the removal of impurities.

A partially purified MAb I affinity (Protein A) captured eluate wasprepared according to Example 5. The eluate of MAb I was adjusted fromapproximately pH 5 to pH 7 with Tris base (2 M) and filtered through a0.22 micron Millipore Express Plus Stericup filter unit. The solution isreferred to herein as the MAb I Protein A eluate.

Nuchar RGC and RGC Nuchar activated carbon are used hereininterchangeably and refer to the grade of powdered activated carbonobtained from MeadWestVaco Corporation, Richmond, Va., USA. In arepresentative experiment, a 7 mm diameter circular portion (5 μL) ofthe ChromaSorb™ membrane and/or 10 mg of RGC Nuchar activated carbonwere added to a 1.5 mL centrifuge tube along with Tris-HCl buffer, 25mM, pH 7 to equilibrate the adsorbent media. The tubes were centrifugedand the supernatant equilibration buffer was removed. Subsequently, a 1mL volume of the MAb I Protein A eluate was added to a 1.5 mL centrifugetube containing the equilibrated adsorbent(s). The adsorbent media andthe eluate were allowed to interact for 18 hours at room temperatureunder gentle rotation. The tubes were then subjected to centrifugationand the supernatant solutions were analyzed for host cell protein (HCP)and IgG concentration.

HCP analysis was performed using a commercially available ELISA kit fromCygnus Technologies, Southport, N.C., USA, catalog number F550,following kit manufacturer's protocol. IgG concentration was measuredusing an Agilent HPLC system equipped with a Poros® A Protein Aanalytical column. Results of one such experiment are summarized inTable V.

The results show that activated carbon alone and in combination with ananion exchange media was unexpectedly effective for the removal ofimpurities from the monoclonal antibody solution. The combination ofactivated carbon and the anion exchange media (ChromaSorb) removed thegreatest amount of impurities with a log reduction value (LRV) of hostcell protein (HCP) of 1.43. The individual activated carbon and anionexchange media had LRVs of HCP of 1.21 and 0.36 respectively. Theactivated carbon alone and in combination with an anion exchange mediaboth had excellent recoveries of the monoclonal antibody product of 95%and 95% respectively.

TABLE V MAb I Recovery HCP HCP LRV of Adsorbent media (mg/mL) MAb I(ng/mL) (ppm) HCP Control (nothing 6.92 NA 1748 252 NA added) ActivatedCarbon 6.58 95% 102 16 1.21 ChromaSorb 6.74 97% 743 110 0.36 ActivatedCarbon 6.58 95% 62 9 1.43 and ChromaSorb NA—Not Applicable

Example 9. Static Soak Treatment of an Affinity Captured Eluate of a MAbwith Activated Carbon and/or an Anion Exchange Media

Activated Carbon Alone and in Combination with an Anion Exchange Mediawas examined for the removal of impurities from an affinity (Protein A)captured monoclonal antibody eluate that contains a different monoclonalantibody than MAb I (referred to as MAb II) to demonstrate thatactivated carbon alone and in combination with an anion exchange mediaprovides a unique and unexpectedly effective method that can be appliedto purification of a variety of different monoclonal antibodies.

A second partially purified MAb II affinity (Protein A) captured eluatewas prepared according to Example 5. The eluate of MAb II was adjustedfrom approximately pH 5 to pH 7 with Tris base (2 M) and filteredthrough a 0.22 micron Millipore Express Plus Stericup filter unit. Thesolution is referred to herein as the MAb II Protein A eluate.

A 7 mm diameter circular portion (5 μL) of the ChromaSorb membraneand/or 10 mg of RGC Nuchar activated carbon were added to a 1.5 mLcentrifuge tube along with Tris-HCl buffer, 25 mM, pH 7 in order toequilibrate the adsorbent media. The tubes were centrifuged and thesupernatant equilibration buffer was removed. Subsequently, 1 mL volumeof the pH adjusted MAb II Protein A eluate was added to a 1.5 mLcentrifuge tube containing the equilibrated adsorbent(s). The adsorbentmedia and the eluate were allowed to interact for 18 hours at roomtemperature under gentle rotation. The tubes were then subjected tocentrifugation and the supernatant solutions were analyzed for host cellprotein (HCP) and IgG concentration. HCP analysis was performed using acommercially available ELISA kit from Cygnus Technologies, Southport,N.C., USA (catalog number F550), following kit manufacturer's protocol.IgG concentration was measured using an Agilent HPLC system equippedwith a Poros® A Protein A analytical column. Results are summarized inTable VI.

The results show that activated carbon alone and in combination with ananion exchange media was unexpectedly effective for the removal ofimpurities from a solution containing a second type of monoclonalantibody. The combination of activated carbon and the anion exchangemedia (ChromaSorb™) removed the greatest amount of impurities with a logreduction value (LRV) of host cell protein (HCP) of 1.78. The individualactivated carbon and anion exchange media had LRVs of HCP of 1.08 and0.64 respectively. The activated carbon alone and in combination with ananion exchange media both had good recoveries of the monoclonal antibodyproduct of 87% and 85% respectively.

TABLE VI MAb II Recovery HCP HCP LRV of Adsorbent media (mg/mL) MAb II(ng/mL) (ppm) HCP Control (nothing 18.19 NA 23418 1287 NA added)Activated Carbon 15.89 87% 4681 295 0.64 ChromaSorb 16.08 88% 1711 1061.08 Activated Carbon 15.44 85% 329 21 1.78 and ChromaSorb NA—NotApplicable

Example 10. Static Soak Treatment of a Non-Affinity Captured Eluate ofMAb I with Activated Carbon and/or an Anion Exchange Media

Activated carbon alone and in combination with an anion exchange mediawas examined for the removal of impurities from a non-affinity (cationexchange) captured monoclonal antibody eluate to demonstrate thatactivated carbon alone and in combination with an anion exchange mediaprovides a unique and unexpectedly effective method for the removal ofimpurities. Relative to affinity (Protein A) captured eluate thenon-affinity (cation exchange) captured eluate contains different typesof impurities at significantly higher levels. The application ofactivated carbon for the purification of a non-affinity captured eluatedemonstrates that this method is general and can be applied to thepurification of a variety of different protein eluates.

In a separate experiment, partially purified MAb I CEX eluate wasprepared as described in Example 6. The eluate was diluted by a factorof 4 with a buffer solution (Tris-HCl buffer, 25 mM, pH 7) and thenfiltered through a 0.22 micron Millipore Express Plus Stericup filterunit. The solution is referred to herein as the MAb I CEX eluate.

Two 10 mm diameter circular portions (19.6 μL) of the ChromaSorbmembrane and/or 20 mg of RGC Nuchar activated carbon were added to a 1.5mL centrifuge tube along with Tris-HCl buffer, 25 mM, pH 7 in order toequilibrate the adsorbent media. The tubes were centrifuged and thesupernatant equilibration buffer was removed. Subsequently, a 1 mLvolume of the MAb I CEX eluate was added to a 1.5 mL centrifuge tubecontaining the equilibrated adsorbent(s). The adsorbent media and theeluate were allowed to interact for 18 hours at room temperature undergentle rotation. The tubes were then subjected to centrifugation and thesupernatant solutions were analyzed for host cell protein (HCP) and IgGconcentration. HCP analysis was performed using a commercially availableELISA kit from Cygnus Technologies, Southport, N.C., USA, catalog numberF550, following kit manufacturer's protocol. IgG concentration wasmeasured using an Agilent HPLC system equipped with a Poros® A Protein Aanalytical column. Results are summarized in Table VII.

The results show that activated carbon alone and in combination with ananion exchange media was unexpectedly effective for the removal ofimpurities from a monoclonal antibody solution that was captured fromcell culture using a non-affinity (cation exchange) chromatography.Non-affinity (cation exchange) based capture media binds more impuritiesalong with the monoclonal antibody than the more specific affinity(Protein A) based capture media. Therefore non-affinity captured eluatecontains different types of impurities at significantly higher levels.The combination of activated carbon and the anion exchange membrane(ChromaSorb™) removed the greatest amount of impurities with a logreduction value (LRV) of host cell protein (HCP) of 1.45. The individualactivated carbon and anion exchange media had LRVs of HCP of 1.20 and0.55 respectively. The activated carbon alone and in combination with ananion exchange media both had good recoveries of the monoclonal antibodyproduct of 95% and 74% respectively.

TABLE VII MAb I recovery HCP HCP LRV of (mg/mL) of MAb I (ng/mL) (ppm)HCP Control 3.14 NA 64,254 20,463 NA activated carbon 2.99 95% 17,0915,716 0.55 ChromaSorb 2.93 93% 3,817 1,303 1.20 activated carbon 2.3274% 1,676 722 1.45 and ChromaSorb NA—Not Applicable

Example 11. Static Soak Treatment of an Affinity Captured Eluate of MAbI with Activated Carbon at Different pH Values

Activated carbon was examined for the removal of impurities from anaffinity (Protein A) captured monoclonal antibody eluate at differentsolution pHs to demonstrate that activated carbon is effective over avariety of different solution conditions.

In a separate experiment, a partially purified MAb I affinity (ProteinA) captured eluate was prepared according to Example 5. The eluate ofMAb I was adjusted from approximately pH 5 to pH 7 with Tris base (2 M)and filtered through a 0.22 micron Millipore Express Plus Stericupfilter unit. The solution is referred to herein as the MAb I Protein Aeluate.

The pH of MAb I Protein A eluate (20 mL) was adjusted to 5, 6, 7, or 8by the addition of Tris base (2 M) or acetic acid (3 M). The resultingpH adjusted MAb I Protein A eluates were subsequently sterile filteredusing Millipore Express® 0.22 micron membrane to remove any cloudiness.RGC Nuchar activated carbon (10 mg) was then added to a 1.5 mLcentrifuge tubes along with Tris-HCl buffer, 25 mM, pH 7 in order toequilibrate the activated carbon. The tubes were centrifuged and thesupernatant equilibration buffer was removed. 1 mL of the pH adjustedMAb I Protein A eluates were subsequently added to the tubes. Theadsorbent media and the eluates were allowed to interact for 18 hours atroom temperature under gentle rotation. The tubes were then subjected tocentrifugation and 0.5 mL of the supernatant was removed and analyzedfor host cell protein (HCP) and IgG concentration. HCP analysis wasperformed using a commercially available ELISA kit from CygnusTechnologies, Southport, N.C., USA, catalog number F550, following kitmanufacturer's protocol. IgG concentration was measured using an AgilentHPLC system equipped with a Poros® A Protein A analytical column.Results are summarized in Table VIII.

The results show that activated carbon was unexpectedly effective forthe removal of impurities from a monoclonal antibody solution over awide pH range. The log reduction value (LRV) of host cell protein (HCP)was very similar for pH 6, pH 7, and pH 8 ranging from 1.27 to 1.30 LRVof HCP. Activated carbon still provided selective impurity removal at pH5 although the LRV of HCP was reduced to 0.70. The activated carbon hadexcellent recoveries of the monoclonal antibody product ranging from 90%to 95% for all the pH conditions examined.

TABLE VIII MAb I (mg/mL) HCP (ng/mL) HCP (ppm) After Recovery afterafter pH control A.C. of MAb I control A.C. control A.C. LRV of HCP 57.31 6.97 95% 5,505 1,048 753 150 0.70 6 7.26 6.88 95% 1,986 101 274 151.27 7 7.04 6.79 96% 1,889 91 268 13 1.30 8 8.63 7.76 90% 1,556 75 18010 1.27

Example 12. Flow-Through Treatment of an Affinity Captured Eluate of MAbI with Activated Carbon and/or Anion Exchange Media

Activated carbon alone and in combination with an anion exchange mediawas examined in a flow-through application for the removal of impuritiesfrom an affinity (Protein A) captured monoclonal antibody eluate todemonstrate that activated carbon alone and in combination with an anionexchange media provides a unique and unexpectedly effective method forthe removal of impurities under flow-through conditions commonly usedfor large scale purification of proteins.

A partially purified MAb I affinity (Protein A) captured eluate wasprepared according to Example 5. The eluate of MAb I was adjusted fromapproximately pH 5 to pH 7 with Tris base (2 M) and filtered through a0.22 micron Millipore Express Plus Stericup filter unit. The solution isreferred to herein as the MAb I Protein A eluate.

Glass Omnifit Chromatography Columns (10 mm diameter, 100 mm length)were loaded with 250 mg of HD Nuchar activated carbon slurried in waterto give a packed column volume of 1 mL. The columns were equilibratedwith buffer solution (Tris-HCl buffer, 25 mM, pH 7). A 0.2 mL ChromaSorbdevice, fabricated as described above in Example 7, was alsoequilibrated with buffer solution (Tris-HCl buffer, 25 mM, pH 7). Threepurification trains were subsequently assembled. The first consisting ofa ChromaSorb device, the second consisting of an activated carboncolumn, and the third consisting of an activated carbon column followedby a ChromaSorb device. 100 mL of the MAb I Protein A eluate was passedthrough each set up at a flow rate of 0.25 mL/min. Ten 10 mL fractionsof the eluate were collected. Pooled samples of all ten as well asselected individual fractions were analyzed for host cell protein (HCP)and IgG concentration. HCP analysis was performed using a commerciallyavailable ELISA kit from Cygnus Technologies, Southport, N.C., USA,catalog number F550, following kit manufacturer's protocol. IgGconcentration was measured using an Agilent HPLC system equipped with aPoros® A Protein A analytical column. Results are summarized in TableIX.

As depicted in FIG. 16 and summarized in Table IX, the results show thatflow-through treatment of the affinity captured eluate with activatedcarbon alone and in combination with an anion exchange media wasunexpectedly effective for the removal of impurities from the monoclonalantibody solution. The combination of activated carbon and the anionexchange membrane (ChromaSorb) removed the greatest amount of impuritieswith a log reduction value (LRV) of host cell protein (HCP) of 1.95. Theindividual activated carbon and anion exchange media had LRVs of HCP of0.96 and 0.23 respectively. The activated carbon alone and incombination with an anion exchange media both had excellent recoveriesof the monoclonal antibody product ranging of 96% and 98% respectively.

TABLE IX MAb I recovery HCP HCP LRV of Flow-through train (mg/mL) of MAbI (ng/mL) (ppm) HCP untreated (control) 9.21 NA 6,259 679 NA ChromaSorbonly 8.92 97% 3,538 397 0.23 Activated Carbon 9.03 98% 1,330 148 0.96only Activated Carbon 8.81 96% 67 8 1.95 followed by ChromaSorb NA—NotApplicable

Example 13. Flow-Through Treatment of an Affinity Captured Eluate of MAbI with Various Anion Exchange Media Alone or after Treatment withActivated Carbon

Activated carbon was examined alone and in combination with a variety ofdifferent commercially available anion exchange media for the removal ofimpurities from an affinity (Protein A) captured monoclonal antibodyeluate to demonstrate activated carbon can be combined with variousdifferent anion exchange media. The commercially available anionexchange media examined included primary amines (ChomaSorb) andquaternary amines (Sartobind Q, Mustang Q, HiTrap Q FF). Thecommercially available anion exchange chemistries were examinedsupported on a membrane (ChomaSorb, Sartobind Q, Mustang Q) and on aresin (HiTrap Q FF).

A partially purified MAb I affinity (Protein A) captured eluate wasprepared according to Example 5. The eluate of MAb I was adjusted fromapproximately pH 5 to pH 7 with Tris base (2 M) and filtered through a0.22 micron Millipore Express Plus Stericup filter unit. The solution isreferred to herein as the MAb I Protein A eluate.

Glass Omnifit Chromatography Columns (10 mm diameter, 100 mm length)were loaded with 250 mg of HD Nuchar activated carbon slurried in waterto give a packed column volume of 1 mL. The columns were equilibratedwith buffer solution (Tris-HCl buffer, 25 mM, pH 7). 3-layer, 0.26 mLSartobind Q membrane devices were manufactured using commerciallyavailable Sartobind Q membrane disks (SIGMA-ALDRICH, St. Louis, Mo.,USA) (0.26 mL, 3 sheets) and the device housing and process used toproduce 0.2 mL ChromaSorb devices fabricated as described above inExample 7. 0.18 mL Acrodisc® Units with Mustang Q membrane werepurchased from Thermo Fisher Scientific, Waltham, Mass., USA. Thesedevices, along with 1 mL HiTrap Q FF prepacked column (GE Healthcare,Pittsburgh, Pa., USA) and 0.2 mL ChromaSorb device, were equilibratedwith buffer solution (Tris-HCl buffer, 25 mM, pH 7).

Nine flow-through purification trains were assembled: a) activatedcarbon, b) ChromaSorb, c) activated carbon followed by ChromaSorb, d)Sartobind Q, e) activated carbon followed by Sartobind Q, f) Mustang Q,g) activated carbon followed by Mustang Q, h) HiTrap Q FF, i) activatedcarbon followed by HiTrap Q FF. 96 mL of the MAb I Protein A eluate wassubsequently passed through each set up at a flow rate of 0.25 mL/min.After passing through the purification trains, the solutions wereanalyzed for host cell protein (HCP) and IgG concentration. HCP analysiswas performed using a commercially available ELISA kit from CygnusTechnologies, Southport, N.C., USA, catalog number F550, following kitmanufacturer's protocol. IgG concentration was measured using an AgilentHPLC system equipped with a Poros® A Protein A analytical column.Results are summarized in Table X.

The results show that the flow-through treatment of an affinity (ProteinA) captured monoclonal antibody eluate with the combination of activatedcarbon with a variety of different anion exchange media was unexpectedlyeffective for the removal of impurities. The log reduction value (LRV)of host cell protein (HCP) was 0.91 for activated carbon alone. Thedifferent commercially available anion exchange media alone had verysimilar LRV of HCP ranging from 0.17 to 0.23. The combination ofactivated carbon followed by the different commercially available anionexchange media had much higher LRV of HCP ranging from 1.70 to 1.93. Thecombination of activated carbon followed by the different anion exchangemedia had excellent recoveries of the monoclonal antibody productranging from 96% to 97%. The data demonstrates that activated carbon isunexpectedly effective for the purification of antibodies in combinationwith a variety of different commercially available anion exchange mediaincluding primary amines (ChomaSorb™) and quaternary amines (SartobindQ, Mustang Q, HiTrap Q FF). The combination of activated carbon washighly effective in combination with commercially available anionexchange chemistries that were supported on a membrane (ChomaSorb,Sartobind Q, Mustang Q) and on a resin (HiTrap Q FF).

TABLE X Recovery MAb I (mg/mL) of MAb I HCP (ng/mL) HCP (ppm) LRV of HCPAEX AEX AEX AEX AEX AEX AEX AEX AEX AEX AEX Media Alone then A.C. alonethen A.C. alone then A.C. alone then A.C. one then A.C. no AEX media NA8.82 NA 96% 5,701 738 618 84 NA 0.91 ChromaSorb 9.20 8.89 100% 96% 3,66070 398 8 0.23 1.93 Sartobind Q 9.26 8.99 100% 97% 4,103 98 443 11 0.191.80 Mustang Q 9.17 8.86  99% 96% 4,001 120 436 14 0.19 1.70 Q Fast Flow9.20 8.76 100% 95% 4,245 81 462 9 0.17 1.86 NA—Not Applicable

Example 14. Flow-Through Treatment of an Affinity Captured Eluate of MAbI with Activated Carbon Followed by Anion Exchange Media or with anAnion Exchange Media Followed by Activated Carbon

The order of activated carbon and an anion exchange media for theflow-through removal of impurities from an affinity (Protein A) capturedmonoclonal antibody eluate was examined to demonstrate that the order ofthe two adsorbers unexpectedly influences their effectiveness. Theexperiment illustrates that the placement of activated carbon before theanion exchange media is important to maximize the ability of thecombination of adsorbers to remove impurities from protein solutions.

A partially purified MAb I affinity (Protein A) captured eluate wasprepared according to Example 5. The eluate of MAb I was adjusted fromapproximately pH 5 to pH 7 with Tris base (2 M) and filtered through a0.22 micron Millipore Express Plus Stericup filter unit. The solution isreferred to herein as the MAb I Protein A eluate.

Glass Omnifit Chromatography Columns (10 mm diameter, 100 mm length)were loaded with 250 mg of HD Nuchar activated carbon slurried in waterto give a packed column volume of 1 mL. The columns were equilibratedwith buffer solution (Tris-HCl buffer, 25 mM, pH 7). A 0.2 mL ChromaSorbmembrane device fabricated as described above in Example 7 was alsoequilibrated with buffer solution (Tris-HCl buffer, 25 mM, pH 7). Twoflow-through trains were assembled. The first had the activated carboncolumn followed by the ChromaSorb membrane device while the second hadthe reverse order with the ChromaSorb membrane device followed by theactivated carbon column. 96 mL of the MAb I Protein A eluate was passedthrough each set up at a flow rate of 0.25 mL/min. After passing throughthe purification trains, the solutions were analyzed for host cellprotein (HCP) and IgG concentration. HCP analysis was performed using acommercially available ELISA kit from Cygnus Technologies, Southport,N.C., USA, catalog number F550, following kit manufacturer's protocol.IgG concentration was measured using an Agilent HPLC system equippedwith a Poros® A Protein A analytical column. Results are summarized inTable XI.

The results show the unexpected result that the order of the activatedcarbon and an anion exchange media (ChromaSorb) are important to theeffectiveness of the flow-through purification of an affinity (ProteinA) captured eluate. The log reduction value (LRV) of host cell protein(HCP) was 1.87 when activated carbon was placed in front of anionexchange media. The LRV of HCP was reduced to 1.38 when the anionexchange media was place in front of the activated carbon. The order ofthe activated carbon and the anion exchange media had no influence onthe recovery of the antibody, which was 97% for both orders of theadsorbers. The results reveal an important understanding that placingthe activated carbon before the anion exchange media is important tomaximize the effectiveness of the combination of two adsorbers to removeimpurities from protein solutions.

TABLE XI MAb I recovery HCP HCP LRV of Flow-through train (mg/mL) of MAbI (ng/mL) (ppm) HCP untreated (control) 9.24 NA 4,986 540 NA ChromaSorb8.95 97% 202 23 1.38 followed by activated carbon activated carbon 8.9597% 65 7 1.87 followed by ChromaSorb NA—Not Applicable

Example 15. Flow-Through Treatment of a Non-Affinity Captured Eluate ofMAb I with Activated Carbon

Activated carbon was examined in a flow-through application for theremoval of impurities from a non-affinity (cation exchange) capturedmonoclonal antibody eluate to demonstrate that activated carbon providesa unique and unexpectedly effective method for the removal ofimpurities. Relative to an affinity (Protein A) captured eluate thenon-affinity (cation exchange) captured eluate contains different typesof impurities at significantly higher levels. The application ofactivated carbon for the purification of a non-affinity captured eluatedemonstrates that this method is general and can be applied to thepurification of a variety of different protein solutions.

A partially purified MAb I CEX eluate was prepared as described inExample 6. The eluate was diluted by a factor of 4 with a buffersolution (Tris-HCl buffer, 25 mM, pH 7). The diluted eluate of MAb I wasadjusted from approximately pH 6 to pH 7 with Tris base (2 M) andfiltered through a 0.22 micron Millipore Express Plus Stericup filterunit. The solution is referred to herein as the MAb I CEX eluate.

Glass Omnifit Chromatography Column (10 mm diameter, 100 mm length) wasloaded with 250 mg of HD Nuchar activated carbon slurried in water togive a packed column volume of 1 mL. The column was equilibrated with abuffer solution (Tris-HCl buffer, 25 mM, pH 7).

70 mL of the MAb I CEX eluate was then passed through the column ofactivated carbon at a flow rate of 0.25 mL/min. Seven 10 mL fractions ofthe eluate were collected. Pooled samples of all seven as well asselected individual fractions were analyzed for host cell protein (HCP)and IgG concentration. HCP analysis was performed using a commerciallyavailable ELISA kit from Cygnus Technologies, Southport, N.C., USA,catalog number F550, following kit manufacturer's protocol. IgGconcentration was measured using an Agilent HPLC system equipped with aPoros® A Protein A analytical column. Results are summarized in TableXI.

As depicted in FIG. 16 and summarized in Table XII below, the resultsshow that flow-through purification with activated carbon wasunexpectedly effective for the removal of impurities from a non-affinity(cation exchange) captured monoclonal antibody eluate. Non-affinity(cation exchange) based capture media binds more impurities along withthe monoclonal antibody than the more specific affinity (Protein A)based capture media. Therefore non-affinity captured eluate containsdifferent types of impurities at significantly higher levels. Activatedcarbon provided a log reduction value (LRV) of host cell protein (HCP)of 0.76 and had 89% recovery of the monoclonal antibody product. Theseresults suggest that activated carbon can be used to remove impuritiesfrom a variety of different protein solutions.

TABLE XII MAb I recovery HCP HCP LRV of Flow-through train (mg/mL) ofMAb I (ng/mL) (ppm) HCP untreated (control) 3.13 NA 66,269 21,172 NAactivated carbon 2.79 89% 10,343 3,707 0.76 column NA—Not Applicable

Example 16. Flow-Through Purification of an Affinity Captured Eluate ofMAb I with Activated Carbon Packed Column and an ActivatedCarbon-Cellulose Device

Activated carbon packed in a column or blended into a cellulose sheetwas examined in a flow-through application for the removal of impuritiesfrom an affinity (Protein A) captured monoclonal antibody eluate todemonstrate that the activated carbon provides a unique and unexpectedlyeffective method for the removal of impurities in different formats.

A partially purified MAb I affinity (Protein A) captured eluate wasprepared according to Example 5. The eluate of MAb I was adjusted fromapproximately pH 5 to pH 7 with Tris base (2 M) and filtered through a0.22 micron Millipore Express Plus Stericup filter unit. The solution isreferred to herein as the MAb I Protein A eluate.

Glass Omnifit Chromatography Columns (15 mm diameter, 100 mm length)were loaded with 600 mg of HD Nuchar activated carbon slurried in waterto give a packed column volume of 2.4 mL. Activated carbon-impregnatedcellulose sheet media, commercially available in a Millistak+Pod CRdevice from Millipore Corporation, Billerica, Mass., was placed in anovermolded polypropylene syringe device, internal filtration area 25 mm,4.6 mL bed volume, equipped with Luer connectors for inlet and outlet.

The column and the activated carbon-cellulose device were equilibratedwith buffer solution (Tris-HCl buffer, 25 mM, pH 7). Two flow-throughtrains were assembled. The first had the activated carbon column whilethe second had the activated carbon-cellulose device. 315 mL of the MAbI Protein A eluate was subsequently passed through each set up, at aflow rate of 0.75 mL/min. After passing through the purification trains,the solutions were analyzed for host cell protein (HCP) and IgGconcentration. HCP analysis was performed using a commercially availableELISA kit from Cygnus Technologies, Southport, N.C., USA (catalog numberF550), following kit manufacturer's protocol. IgG concentration wasmeasured using an Agilent HPLC system equipped with a Poros® A Protein Aanalytical column. Results are summarized in Table XIII.

The results show that the flow-through purification with activatedcarbon was unexpectedly effective for the removal of impurities from anaffinity (Protein A) captured monoclonal antibody eluate when packedinto column or blended into a cellulose sheet. Activated carbon packedinto a column or blended into a cellulose sheet both gave very similarlog reduction values (LRV) of host cell protein (HCP) of 0.95 and 0.97respectively. They also had very similar recoveries of the monoclonalantibody product of 91% for the column and 87% for the cellulose sheet.These results suggest that activated carbon can be used effectively forthe removal of impurities from protein solutions when blended into acellulose sheet.

TABLE XIII MAb I recovery HCP HCP LRV of Flow-through train (mg/mL) ofMAb I (ng/mL) (ppm) HCP untreated (control) 9.79 NA 6,229 642 NA HDNuchar 8.92 91% 682 76 0.95 activated carbon Column activated 8.53 87%620 73 0.97 carbon-cellulose device NA—Not Applicable

Example 17. Flow-Through Purification of an Affinity Captured Eluate ofMAb I with Two Other Types of Activated Carbon

Two other types of commercially available activated carbon were examinedin a flow-through application for the removal of impurities from anaffinity (Protein A) captured monoclonal antibody eluate to demonstratethe unexpected result that a variety of different activated carbons canbe used for the removal of impurities from protein solutions.

A partially purified MAb I affinity (Protein A) captured eluate wasprepared according to Example 5. The eluate of MAb I was adjusted fromapproximately pH 5 to pH 7 with Tris base (2 M) and filtered through a0.22 micron Millipore Express Plus Stericup filter unit. The solution isreferred to herein as the MAb I Protein A eluate.

Glass Omnifit Chromatography Columns (5 mm diameter, 100 mm length) wereloaded with 125 mg of Chemviron Pulsorb PGC activated carbon (ChemvironCarbon, Feluy, Belgium) or Norit A Supra USP activated carbon (NoritAmericas Inc., Marshall, Tex., USA) slurried in water to give a packedcolumn volume of 0.24 mL. The columns were equilibrated with buffersolution (Tris-HCl buffer, 25 mM, pH 7). Two flow-through trains wereassembled. The first had the Chemviron Pulsorb PGC activated carboncolumn while the second had the Norit A Supra USP activated carboncolumn. 96 mL of the MAb I Protein A eluate was passed through each setup at a flow rate of 0.25 mL/min. After passing through the purificationtrains, the solutions were analyzed for host cell protein (HCP) and IgGconcentration. HCP analysis was performed using a commercially availableELISA kit from Cygnus Technologies, Southport, N.C., USA (catalog numberF550), following the kit manufacturer's protocol. IgG concentration wasmeasured using an Agilent HPLC system equipped with a Poros® A Protein Aanalytical column. Results are summarized in Table XIV.

The results show that the flow-through purification with two other typesof activated carbon were unexpectedly effective for the removal ofimpurities from an affinity (Protein A) captured monoclonal antibodyeluate. The Chemviron Pulsorb PSG and Norit A Supra USP both removedimpurities with log reduction values (LRV) of host cell protein (HCP) of0.40 and 0.48 respectively. They also had excellent recoveries of themonoclonal antibody product of 100% for Chemviron Pulsorb PSG and 100%for Norit A Supra USP. These results suggest that several differenttypes of activated carbon can be used for the removal of impurities fromprotein solutions.

TABLE XIV MAb I recovery HCP HCP LRV of Flow-through train (mg/mL) ofMAb (ng/mL) (ppm) HCP untreated (control) 7.15 NA 1026 144 NA ChemvironPulsorb 7.18 100% 409 57 0.40 PGC Norit A Supra USP 7.17 100% 341 480.48 NA—Not Applicable

Example 18. Flow-Through Purification of an Affinity Captured Eluate ofMAb I in the Presence of Different Buffer Salts

Activated carbon was examined in a flow-through application for theremoval of impurities from an affinity (Protein A) captured monoclonalantibody eluate with various different salts added to demonstrate thatactivated carbon provides a unique and unexpectedly effective method forthe removal of impurities in the presence of many different salts. Theinvestigation illustrates that this method is general and can be appliedto the purification of proteins in a variety of different buffer salts.

A partially purified MAb I affinity (Protein A) captured eluate wasprepared according to Example 5. The eluate of MAb I was adjusted fromapproximately pH 5 to pH 7 with Tris base (2 M) and filtered through a0.22 micron Millipore Express Plus Stericup filter unit. The solution isreferred to herein as the MAb I Protein A eluate.

To a 50 mL portion of the MAb I Protein A eluate, 10 mL aqueous solutioncontaining of 300 mM of various salts was added, where the salts wereammonium sulfate, ethylenediaminetetraacetic acid disodium saltdehydrate (EDTA), 2-(N-morpholino)ethanesulfonic acid (MES), sodiumchloride, Trisodium citrate dehydrate, sodium phosphate dibasicheptahydrate, and Trizma® Pre-set crystals, pH 7.0 (Tris-HCl).

A solution diluted with 10 mL water was used as a control. The pH of thesalt spiked Protein A eluate were adjusted back to 7 with 2 M Tris baseor 3 M acetic acid. The solution is referred to herein as the saltspiked MAb I Protein A eluate.

Glass Omnifit Chromatography Columns (5 mm diameter, 100 mm length) wereloaded with 125 mg of HD Nuchar activated carbon slurried in water togive a packed column volume of 0.5 mL. The columns were equilibratedwith buffer solution (Tris-HCl buffer, 25 mM, pH 7). 40 mL of the saltspiked MAb I Protein A eluate was then passed through the columns ofactivated carbon at a flow rate of 0.125 mL/min. After passing throughthe columns, the solutions were analyzed for host cell protein (HCP) andIgG concentration. HCP analysis was performed using a commerciallyavailable ELISA kit from Cygnus Technologies, Southport, N.C., USA(catalog number F550), following the kit manufacturer's protocol. IgGconcentration was measured using an Agilent HPLC system equipped with aPoros® A Protein A analytical column. Results are summarized in TableXV.

The results show that the flow-through purification of an affinity(Protein A) captured monoclonal antibody eluate with activated carbonwas unexpectedly effective for the removal of impurities in the presenceof a variety of different salt additives. The activated carbon removedimpurities in the presence of all the added salts with log reductionvalues (LRV) of host cell protein (HCP) ranging from 0.63 to 1.00. Theyalso had excellent recoveries of the monoclonal antibody product rangingfrom 92% to 96%. These results suggest that activated carbon can be usedfor the removal of impurities from protein solutions in a variety ofdifferent salt buffers.

TABLE XV MAb I (mg/mL) HCP (ng/mL) HCP (ppm) before after Recoverybefore after before after spiked salt A.C. A.C. of MAb I A.C. A.C. A.C.A.C. LRV of HCP water only (control) 7.33 6.86 94% 987 76 135 11 1.08Tris 6.99 6.68 96% 1,194 119 171 18 0.98 sodium chloride 6.58 6.33 96%1,151 111 175 17 1.00 ammonium sulfate 6.93 6.66 96% 1,359 177 196 270.87 disodium phosphate 6.92 6.59 95% 1,293 153 187 23 0.90 sodiumcitrate 6.53 6.03 92% 1,351 241 207 40 0.71 EDTA 6.71 6.39 95% 1,304 293194 46 0.63 MES 6.90 6.54 94% 1,110 132 161 20 0.90

Example 19. Flow-Through Purification of Protein A Eluate at pH 5 and pH7

Activated carbon was examined in a flow-through application for theremoval of impurities from an affinity (Protein A) captured monoclonalantibody eluate at two different pH conditions to demonstrate thatactivated carbon provides a unique and unexpectedly effective method forthe flow-through removal of impurities at different solution pHconditions. The investigation illustrates that this method is generaland can be applied to the purification of proteins under different pHconditions.

A partially purified MAb I affinity (Protein A) captured eluate wasprepared according to Example 5. The solution is referred to herein asthe pH 5 MAb I Protein A eluate.

A portion of the eluate of MAb I prepared according to Example 5 wasadjusted from approximately pH 5 to pH 7 with Tris base (2 M) andfiltered through a 0.22 micron Millipore Express Plus Stericup filterunit. The solution is referred to herein as the pH 7 MAb I Protein Aeluate.

Glass Omnifit Chromatography Columns (15 mm diameter, 100 mm length)were loaded with 1.25 g of HD Nuchar activated carbon slurried in waterto give a packed column volume of 5 mL. The columns were equilibratedwith buffer solution (Tris-HCl buffer, 25 mM, pH 7). Then 500 mL of thepH 5 MAb I Protein A eluate or the pH 7 MAb I Protein A eluate waspassed through the activated column at a flow rate of 1.25 mL/min. Afterpassing through the purification trains, the solutions were analyzed forhost cell protein (HCP) and IgG concentration. HCP analysis wasperformed using a commercially available ELISA kit from CygnusTechnologies, Southport, N.C., USA (catalog number F550), following thekit manufacturer's instructions. IgG concentration was measured using anAgilent HPLC system equipped with a Poros® A Protein A analyticalcolumn. Results are summarized in Table XVI.

The results show that the flow-through purification of an affinity(Protein A) captured monoclonal antibody eluate with activated carbonwas unexpectedly effective for the removal of impurities at both pH 5and pH 7. The activated carbon removed impurities with log reductionvalues (LRV) of host cell protein (HCP) of 0.85 at pH 5 and 1.15 at pH7. Activated carbon also had excellent recoveries of the monoclonalantibody with 97% at pH 5 and 101% at pH 7. These results suggest thatactivated carbon can be used for the removal of impurities for proteinsolutions at different pH conditions.

TABLE XVI MAb I (mg/mL) HCP (ng/mL) HCP (ppm) before after Recoverybefore after before after pH A.C. A.C. of MAb I A.C. A.C. A.C. A.C. LRVof HCP 5 9.22 8.98  97% 3,429 486 371 54 0.85 7 8.61 8.68 101% 1,774 124206 14 1.15

Example 20. Flow-Through Purification of a Protein A Eluate PreparedUsing Continuous Chromatography

This representative experiment demonstrates that Activated Carbon and ananion-exchange chromatography device can be used to purify a Protein Aeluate obtained using Continuous multicolumn chromatography (CMC).

In this example, a monoclonal antibody (MAb II) is purified using athree column continuous multicolumn chromatography (CMC) method usingProsep® Ultra Plus Protein A resin, as described in co-pending EuropeanPatent Application No. EP12002828.7, incorporated by reference herein.The Protein A eluate is pooled and processed through an activated carbondevice followed by an anion exchange chromatography device (i.e.,ChromaSorb™), as described in Example 12.

As demonstrated in FIG. 18 , successful purification of the monoclonalantibody is obtained, as measured by the reduction of HCP concentrationbelow 10 ppm, when a combination of activated carbon and an anionexchange chromatography device are used.

Example 21. Connecting Several Flow-Through Impurity Removal Steps

In this representative experiment, the feasibility of connecting severalimpurity removal steps in flow-through mode to operate as a single unitoperation or process step is demonstrated, while meeting product purityand yield targets.

In this example, individual devices, namely, an activated carbon device,an anion exchange chromatography device (i.e., ChromaSorb™), a cationexchange chromatography device and a virus filtration device (i.e.,Viresolve® Pro) are connected to operate in a flow-through mode.Further, an in-line static mixer and/or a surge tank are positionedbetween the anion exchange chromatography and the cation exchangechromatography devices, in order to achieve a pH change. Lastly, anoptional depth filter is positioned upstream of the activated carbondevice, in case the sample being purified is turbid.

FIG. 19 depicts a schematic representation of the experimental set up toperform a flow-through purification process step, which includes thefollowing described devices.

Additionally, the necessary pumps, valves, sensors etc. may also beincluded in such a set up.

All devices are individually wetted at a different station, and thenassembled. The devices are wetted and pre-treated according to themanufacturer's protocol. Briefly, the depth filter (A1HC grade) isflushed with 100 L/m² of water followed by 5 volumes of equilibrationbuffer 1 (EB1; Protein A elution buffer adjusted to pH 7.5 with 1 MTris-base, pH 11). 2.5 mL of activated carbon is packed into a 2.5 cmOmnifit column as described in Example 12, to produce antibody loadingof 0.55 kg/L. The column is flushed with 10 CV water, and thenequilibrated with EB1 until the pH is stabilized to pH 7.5. TwoChromaSorb devices (0.2 and 0.12 mL) are connected in series to get aloading of 4.3 kg/L. The devices are wetted with water at 12.5 CV/minfor at least 10 min, followed by 5 DV (Device Volumes) EB1. A disposablehelical static mixer (Koflo Corporation, Cary, Ill.) with 12 elements isused to perform in-line pH adjustments. Two 1.2 mL cation-exchangeflow-through devices for aggregate removal are connected in parallel toremove aggregates. The MAb loading on the CEX devices is about 570mg/mL. These devices are wetted with 10 DV water, followed by 5 DVequilibration buffer 2 (EB2; EB1 Adjusted to pH 5.0 using 1 M aceticacid). The devices are further treated with 5 DV (device volumes) ofEB2+1 M NaCl, and then equilibrated with 5 DV EB2. A 3.1 cm² VireSolve®Pro device is wetted with water pressurized at 30 psi for at least 10min. The flow rate is then monitored every minute until the flow rateremains constant for 3 consecutive minutes. After all the devices arewetted and equilibrated, they are connected to as shown in FIG. 19 . EB1is run through the entire system until all pressure readings, and pHreadings are stabilized. Following equilibration, the feed is passedthrough the flow-through train. During the run, samples are collectedbefore the surge tank and after Viresolve® Pro to monitor IgGconcentration and impurity levels (HCP, DNA, leached PrA andaggregates). After the feed is processed, the system is flushed with 3dead volumes of EB1 to recover protein in the devices and in theplumbing.

The feed for the connected flow-through process is protein A eluate ofMAb II, produced in a batch protein A process. The natural level ofaggregates in this MAb does not exceed 1%, so a special procedure wasdeveloped to increase the level of aggregates. The solution pH is raisedto 11 with aqueous NaOH, with gentle stirring, and held for 1 hour. ThepH is then lowered slowly to pH 5 with aqueous HCl under gentlestirring. The pH cycle is repeated 4 more times. The final level ofaggregates is about 5%, mostly consisting of MAb dimers and trimers asmeasured by SEC. The feed is then dialyzed into Tris-HCl buffer, pH 7.5,conductivity about 3 mS/cm.

The amount of MAb feed processed for this run is 102 mL of 13.5 mg/mLMAb at a flow rate of 0.6 mL/min.

The HCP breakthrough as a function of time after ChromaSorb™ is belowthe upper limit of 10 ppm (FIG. 20 ). The aggregates are reduced from 5%to 1.1% by the CEX device (FIG. 21 ). The MAb II yield of the connectedprocess is 92%. The throughput on Viresolve® Pro device is >3.7 kg/m².

Accordingly, the foregoing Example demonstrates that several devices canbe connected to operate in a flow-through mode successfully, thereby toachieve the desired product purity and yield targets.

Example 22. Connecting Flow-Through Purification Process Step withContinuous Bind and Elute Chromatography Capture Step

In this representative experiment, a flow-through purification process,as described herein, was directly linked to a continuous bind and elutechromatography capture process, which precedes the flow-throughpurification.

In this example, a CHO-based monoclonal antibody (MAbII) is produced ina fed-batch bioreactor. A total of 7 L of cell culture is contacted witha solution of stimulus responsive polymer to make a final stimulusresponsive polymer concentration of 0.2% v/v. The cell culture isallowed to mix for approximately 10 minutes. 175 mL of 2 M K₂HPO₄solution is added and allowed to mix for an additional 10 minutes. ThepH is then raised to 7.0 with 2 M tris base and allowed to mix for 15minutes. The solution is then centrifuged in 2 L aliquots at 4,500×g for10 minutes and the supernatant is decanted and retained. The solids aredisposed off. The cell culture supernatant is pooled and then mixed with5 M NaCl at a 1:10 ratio in a batch mode with continuous stirring. Thefinal conductivity of the solution is measured at this point and is at55±5 mS/cm. The resulting higher NaCl concentration solution is sterilefiltered through a 0.22 μm Express filter. The sterile filtered solutionis the loading material for the Protein A chromatography.

The protein A capture step consists of two protein A columns runningwith a method on a modified Akta Explorer 100. The Protein A columnshave 10 mL of ProSep® Ultra Plus protein A media packed into 1.6 cm IDVantage-L (EMD Millipore) chromatography columns to bed heights of 10.25and 10.85 cm. The columns are equilibrated with 1×TBS, 0.5 M NaCl for 5column volumes, CVs (all column volumes are based on the smallestcolumn). Throughout the run, the loading flow rate is set so as to havea loading residence time of about one minute. During the initialloading, both columns are placed in series, where the effluent of theprimary column is loaded directly onto the secondary column until aspecific load volume is reached. After a specific loading volume ispassed over the columns, the feed is stopped and two CVs of theequilibration buffer is passed through the primary column to thesecondary column. The primary column is then positioned to undergowashing, elution, cleaning and reequilibration, while the secondarycolumn is loaded as the primary column. Following the reequilibration ofthe first column, that column is then moved to the secondary position toreside in series with the now primary column. This series of events isrepeated with each column taking the primary position after the originalprimary position column is loaded to a set volume. Each column is loadeda total of seven times. The elutions from each column are collected witha fraction collector, using a UV trigger to control the start time ofthe elution and collected to a constant volume of approximately 3.5 CVs.

The flow-through purification train consists of six main devices:optional depth filter (for precipitate removal after pH adjustment to pH7.5); activated carbon; ChromaSorb™; static mixer and/or surge tank forin-line pH adjustment; cation-exchange flow-through device for aggregateremoval (CEX device); and virus filtration device (i.e., Viresolve®Pro).

FIG. 19 illustrates the order in which these devices are connected.

All devices are individually wetted at a different station, and thenassembled as shown in FIG. 19 . The devices are wetted and pre-treatedaccording to the manufacturer's protocol or as described earlier.Briefly, the depth filter (A1HC) is flushed with 100 L/m² of waterfollowed by 5 volumes of equilibration buffer 1 (EB1; PrA elution bufferadjusted to pH 7.5 with 1 M Tris-base, pH 11). 10 mL of activated carbonis packed into a 2.5 cm Omnifit column as described in Example 12. Thecolumn is flushed with 10 CV water, and then equilibrated with EB1 untilthe pH is stabilized to pH 7.5. 1.2 mL of ChromaSorb membrane (7 layers)is stacked into a 47 mm diameter Swinex device. The device is wettedwith water at 12.5 CV/min for at least 10 min, followed by 5 devicevolumes (DVs) of EB1. A disposable helical static mixer (KofloCorporation, Cary, Ill.) with 12 elements is used to perform in-line pHadjustments. A 3-layer cation-exchange chromatography device (0.12 mLmembrane volume) is wetted with 10 DVs water, followed by 5 DVs ofequilibration buffer 2 (EB2: EB1 adjusted to pH 5.0 using 1 M aceticacid). The device is further treated with 5 DVs of EB2+1 M NaCl, andthen equilibrated with 5 DV EB2. A 3.1 cm2 Viresolve® Pro virusfiltration device is wetted with water pressurized at 30 psi for atleast 10 minutes. The flow rate is then monitored every minute until theflow rate remains constant for 3 consecutive minutes. After all thedevices are wetted and equilibrated, they are connected as shown in FIG.19 . EB1 is run through the entire system until all pressure readings,and pH readings are stabilized. Following equilibration, feed (PrAelution adjusted to pH 7.5) is passed through the Flow-ThroughPurification train. During the run, samples are collected before thesurge tank and after Viresolve® Pro to monitor IgG concentration andimpurity levels (HCP, DNA, leached PrA and aggregates). After the feedis processed, the system is flushed with 3 dead volumes of EB1 torecover protein in the devices and in the plumbing.

FIG. 22 shows the pressure readings before depth filter, activatedcarbon, and ViresolvePro. The pressure on the depth filter remainsunchanged for most part of the run, but rises towards the end suggestingsome precipitation of the Protein A elution feed fractions towards theend of the Protein A runs. The activated carbon column remains fairlyprotected from any precipitate due the depth filter upstream of theactivated carbon. The ViresolvePro pressure rises slowly with time, butis well below the operating maximum limit (50 psi).

The final HCP in the ViresolvePro pool is <1 ppm (Table XVI). Theaverage leached Protein A in the elution fractions is 32 ppm. Theleached Protein A in the Viresolve® Pro pool is 4 ppm. The aggregatesare reduced from 1% to 0.4%. Table XVII below depicts the results of anexperiment to investigate flow-through purification performance whenconnected with a continuous bind and elute chromatography process step.

TABLE XVII Flow-Through Purification Yield (%) 97.8% Average HCP fromall PrA elutions to  172 → 1.75 ViresolvePro pool (ppm) Aggregates inViresolvePro pool (%)    1 → 0.4% Leached PrA in ViresolvePro pool (ppm)32 → 4  ViresolvePro throughput (kg/m²) >6.1 Dilution factorpost-Protein A 1.15x

Example 23. Removal of a Cell Culture Component Impurity Using ActivatedCarbon

In this representative experiment, it is demonstrated that potentialimpurities from cell culture that may persist through the Protein Aaffinity capture step are removed by activated carbon.

A common component of the cell culture media, Insulin, a growthstimulator of mammalian cells, is typically present in the cell culturemedia in concentrations 1-20 mg/L. Recombinant Human Insulin(Incelligent AF from EMD Millipore Corp.) is dissolved in 50 mM Tris pH7.0 buffer at 1 mg/mL, monoclonal antibody MAb II was added toconcentration 7 g/L. A glass Omnifit column is packed with HD Nucharactivated carbon. The solution is flowed through the column at aconstant rate of 0.25 CV/min, to a total MAb II loading of 1 kg/L. Theflow-through pool is analyzed for insulin and antibody concentration.For analysis, an Agilent HPLC system equipped with HC18 column (Cadenza)is used; solvent A: 0.1% TFA in water; solvent B 0.1% TFA inAcetonitrile; optimized gradient of 5%-30% B over 15 minutes was used todetect insulin by UV A214 absorbance. First, a calibration curve iscreated using standard solutions of insulin in the presence of antibody.No insulin is detected in the effluent from the activated carbon column,indicating that the activated carbon has capacity for Insulin in thepresence of MAb, in excess of 240 mg/g.

Example 24. Flow-Through Removal of Impurities from a Turbid Solution ofAffinity Captured Eluate of MAb II with Activated Carbon Cellulose Media

In this representative experiment, it was demonstrated that activatedcarbon is unique and unexpectedly effective for the removal of HCPderived from microbial feeds. A solution of E. coli lysate was spikedwith mAb monoclonal antibody at 1.5 mg/mL. The spiked feed was treatedwith activated carbon packed in a column under flow-through conditions.

Cells from a culture of E. coli were recovered by centrifugation. Thesupernatant was decanted off and the remaining cell pellet was suspendedin a lysis buffer (25 mM Tris, 0.1 mM EDTA at pH 7) by vigorous shakingand stirring. Then a 0.4 mL portion of a stock solution of 100 mM PMSFin ethanol was added. The suspension was split into fractions (˜100 mLeach) and subjected to sonication with 3 sec on and 4 sec off for 5 min.Following sonication the material was pooled and stored at −80 degreesC. for 48 hour. Then the solution was thawed and centrifuged at 4,500×gfor 2 hours to remove the lysed cells. The supernatant was filtered witha Stericup-HV 0.45 μm Durapore membrane (1000 mL, catalogue number:SCHVU11RE, Millipore Corp. Billerica, Mass., 01821, USA). Then it wasfiltered through Stericup-GP 0.22 μm Millipore Express PLUS membrane(1000 mL, catalogue number: SCGPU11RE, Millipore Corp. Billerica, Mass.,01821, USA). The filtered lysate was than combined with 7.5 mL of 10mg/mL of a mAb solution to give a solution spiked with 1.5 mL of themAb. The pH of the solution was measure to be 7.7.

A glass Omnifit Chromatography Column (10 mm diameter, 100 mm length)was loaded with 250 mg of Nuchar HD activated carbon (MeadWestVacoCorporation, Richmond, Va., USA) slurried in water to give a packedcolumn volume of 1 mL. The column was equilibrated with 25 mM Trisbuffer at pH 7. Then 42 mL of the mAb spiked E coli lysate was passedthrough the activated carbon column at a flow rate of 0.25 mL/min givinga residence time of 4 minutes in the activated carbon. The control and apool sample were submitted for analysis for host cell protein (HCP) andIgG concentration.

HCP analysis was performed using a commercially available E. Coli HCPELISA kit from Cygnus Technologies, Southport, N.C., USA (catalog numberF410), following the kit manufacturer's instructions. IgG concentrationwas measured using an Agilent HPLC system equipped with a Poros® AProtein A analytical column. The results show that activated carbon wasunexpectedly effective for the selective removal of HCP derived from amicrobial feed. The concentration of HCP in the lysate was reduced from206,000 ng/mL (133.00 ppm) to 119,000 ng/mL (77,800 ppm) while therecovery of the mAb was 99% starting at a concentration of 1.55 g/L andrecovered at 1.53 g/L. This example demonstrates that activated carboncan be used to remove HCP from non-mammalian cells.

Example 25. Removal of Impurities from a Turbid Solution of AffinityCaptured Eluate of MAb II with Activated Carbon-Cellulose Media and anAnion Exchange Media

In this experiment, it was demonstrated that activated carbon alone andin combination with an ion exchange media is unique and unexpectedlyeffective for the removal of HCP derived from microbial feeds after acapture step.

A solution of E. coli lysate was spiked with mAb monoclonal antibody at1.5 mg/mL and then captured with Protein A chromatography. The capturedfeed was treated with activated carbon packed in a column underflow-through conditions and then by a ChromaSorb™ AEX membrane.

Cells from a culture of E. coli were recovered by centrifugation. Thesupernatant was decanted off and the remaining cell pellet was suspendedin a lysis buffer (25 mM Tris, 0.1 mM EDTA at pH 7) by vigorous shakingand stirring. Then a 0.4 mL portion of a stock solution of 100 mM PMSFin ethanol was added. The suspension was split into fractions (˜100 mLeach) and subjected to sonication with 3 sec on and 4 sec off for 5 min.Following sonication the material was pooled and stored at −80 degreesC. for 48 hour. Then the solution was thawed and centrifuged at 4,500×gfor 2 hours to remove the lysed cells. The supernatant was filtered witha Stericup-HV 0.45 μm Durapore membrane (1000 mL, catalogue number:SCHVU11RE, Millipore Corp. Billerica, Mass., 01821, USA). Then it wasfiltered through Stericup-GP 0.22 μm Millipore Express PLUS membrane(1000 mL, catalogue number: SCGPU11RE, Millipore Corp. Billerica, Mass.,01821, USA). The filtered lysate was than combined with 7.5 mL of 10mg/mL of a mAb solution to give a solution spiked with 1.5 mL of themAb. The pH of the solution was measure to be 7.7. The mAb in the spikedlysate was then captured with Protein A chromatography. Then 30 mL ofthe captured mAb was eluted at a low pH. The elution pH was raised from3-4 to 7 by the drop wise addition of 1 M Tris and then filtered throughStericup-GP 0.22 μm Millipore Express PLUS membrane (250 mL, cataloguenumber: SCGPUO2RE, Millipore Corp. Billerica, Mass., 01821, USA). 1 mLof the pH adjusted elution was set aside for analysis.

A glass Omnifit Chromatography Column (10 mm diameter, 100 mm length)was loaded with 250 mg of Nuchar HD activated carbon (MeadWestVacoCorporation, Richmond, Va., USA) slurried in water to give a packedcolumn volume of 1 mL. The column was equilibrated with 25 mM Trisbuffer at pH 7. Then 29 mL of the mAb Protein A chromatography elutionwas passed through the activated carbon at 0.2 mL/min giving a residencetime of 4 min. 1 mL of the activated carbon treated elution was setaside for analysis.

Then a 0.08 mL ChromaSorb™ device (EMD Millipore Corp. Billerica, Mass.,01821, USA) was wet according to instructions with water then flushedwith flushed with 25 mM Tris pH 7. Then 27 mL of the activated carbontreated elution was passed through the ChromaSorb device at a flow rateof 0.5 mL/min giving of a residence time of 0.16 minutes. 1 mL of theactivated carbon and ChromaSorb treated elution was set aside foranalysis.

Each sample was analyzed for host cell protein (HCP) and IgGconcentration. HCP analysis was performed using a commercially availableE. Coli HCP ELISA kit from Cygnus Technologies, Southport, N.C., USA(catalog number F410), following the kit manufacturer's instructions.IgG concentration was measured using an Agilent HPLC system equippedwith a Poros® A Protein A analytical column.

The results show that activated carbon alone and in combination with anAEX membrane was unexpectedly effective for the selective removal of HCPfrom captured elution of a microbial feed. The concentration of HCP inthe elution was reduced by the activated carbon from 86 ng/mL (7 ppm) to8 ng/mL (0.7 ppm) while the recovery of the mAb was 97% starting at aconcentration of 11.5 g/L and recovered at 11.2 g/L. The concentrationof HCP in the activated carbon treated elution was further reduced bythe ChromaSorb AEX membrane to 3 ng/mL (0.3 ppm) with a 96% overallrecovery of the mAb to give a final concentration of 11.1 g/L. Thisexample demonstrates that activated carbon alone and in combination withan AEX media can be used to remove HCP derived from non-mammalian cellsafter a capture step.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification which arehereby incorporated by reference. The embodiments within thespecification provide an illustration of embodiments in this inventionand should not be construed to limit its scope. The skilled artisanreadily recognizes that many other embodiments are encompassed by thisinvention. All publications and inventions are incorporated by referencein their entirety. To the extent that the material incorporated byreference contradicts or is inconsistent with the present specification,the present specification will supercede any such material. The citationof any references herein is not an admission that such references areprior art to the present invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, cell culture, treatment conditions, and so forth used inthe specification, including claims, are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessotherwise indicated to the contrary, the numerical parameters areapproximations and may vary depending upon the desired properties soughtto be obtained by the present invention. Unless otherwise indicated, theterm “at least” preceding a series of elements is to be understood torefer to every element in the series. Those skilled in the art willrecognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed by the following claims.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only, with a true scope and spirit of the invention beingindicated by the following claims.

What is claimed is:
 1. A purification process for purifying a protein ofinterest from a Protein A eluate comprising a protein of interest andimpurities, the process comprising contacting the eluate with acarbonaceous material and one or more matrices selected from anionexchange media, cation exchange media, and virus filtration media. 2.The method of claim 1, wherein the protein of interest is an antibody ora functional fragment thereof.
 3. The method of claim 1, whereinimpurities in the Protein A eluate comprise host cell proteins (HCPs)and/or DNA.
 4. The method of claim 3, wherein the impurities are HCPsand the level of HCPs after performing the process of claim 3 are lessthan 30% of the level in the eluate.
 5. The method of claim 1, whereinthe process is performed in a flow-through mode.
 6. The method of claim1, wherein the carbonaceous material comprises activated carbon.
 7. Themethod of claim 1, wherein the carbonaceous material comprises activatedcharcoal.
 8. The method of claim 1, wherein the carbonaceous material ispacked in a column, a cartridge or a capsule.
 9. The method of claim 1,wherein the carbonaceous material is impregnated into a porous material.10. The method of claim 1, wherein the carbonaceous material is mixedwith one or more media selected from the group consisting of affinitymedia, anion exchange chromatography (AEX) media, cation exchangechromatography (CEX) media, hydrophobic interaction chromatography (HIC)media and mixed-mode media.